Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec;8(6):1045-1059.
doi: 10.1089/can.2023.0150. Epub 2023 Oct 20.

A Sexually Dimorphic Role for Intestinal Cannabinoid Receptor Subtype-1 in the Behavioral Expression of Anxiety

Courtney P Wood  1 , Bryant Avalos  1 , Camila Alvarez  1 , Nicholas V DiPatrizio  1   2
Affiliations

A Sexually Dimorphic Role for Intestinal Cannabinoid Receptor Subtype-1 in the Behavioral Expression of Anxiety

Courtney P Wood et al. Cannabis Cannabinoid Res. 2023 Dec.

Abstract

Background: Increasing evidence suggests that the endocannabinoid system (ECS) in the brain controls anxiety and may be a therapeutic target for the treatment of anxiety disorders. For example, both pharmacological and genetic disruption of cannabinoid receptor subtype-1 (CB1R) signaling in the central nervous system is associated with increased anxiety-like behaviors in rodents, while activating the system is anxiolytic. Sex is also a critical factor that controls the behavioral expression of anxiety; however, roles for the ECS in the gut in these processes and possible differences between sexes are largely unknown. Objective: In this study, we aimed to determine if CB1Rs in the intestinal epithelium exert control over anxiety-like behaviors in a sex-dependent manner. Methods: We subjected male and female mice with conditional deletion of CB1Rs in the intestinal epithelium (intCB1-/-) and controls (intCB1+/+) to the elevated plus maze (EPM), light/dark box, and open field test. Corticosterone (CORT) levels in plasma were measured at baseline and immediately after EPM exposure. Results: When compared with intCB1+/+ male mice, intCB1-/- male mice exhibited reduced levels of anxiety-like behaviors in the EPM and light/dark box. In contrast to male mice, no differences were found between female intCB1+/+ and intCB1-/- mice. Circulating CORT was higher in female versus male mice for both genotype groups at baseline and after EPM exposure; however, there was no effect of genotype on CORT levels. Conclusions: Collectively, these results indicate that genetic deletion of CB1Rs in the intestinal epithelium is associated with an anxiolytic phenotype in a sex-dependent manner.

Keywords: animal behavior; anxiety; cannabinoid receptors; intestinal epithelium; sex differences.

PubMed Disclaimer

Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
Male intCB1−/− mice exhibit anxiolytic behaviors in the EPM. Male intCB1−/− mice and intCB1+/+ controls were allowed to freely explore the EPM for 5 min. Merged heatmaps of all trials for (A) intCB1+/+ and (B) intCB1−/− mice show general exploration patterns of the open (vertical) and closed (horizontal) arms. Increasing time spent in area designated from blue to red, with red being most time. (C) IntCB1−/− male mice entered the open arms significantly more than controls [t(20)=2.602, p=0.0170]. (D) There were no differences in closed-arm entries between genotypes [t(20)=1.275, p=0.2170]. (E) IntCB1−/− male mice spent more time exploring the open arms when compared with controls [t(20)=3.570, p=0.0019], but there were no differences in (F) cumulative time of closed-arm exploration [t(20)=0.5128, p=0.6137]. (G) IntCB1−/− male mice exhibited an increased number of head dips compared with controls [t(20)=2.736, p=0.0127] and (H) spent more time performing the head dipping behavior than controls [t(20)=3.566, p=0.0019]. All analyses are unpaired Student's t-tests. Data presented as mean±SEM, n=11 mice per genotype. *p<0.05, **p<0.01. EPM, elevated plus maze; SEM, standard error of the mean.
FIG. 2.
FIG. 2.
Female intCB1−/− mice do not perform differently from controls in the EPM. Female intCB1−/− mice and intCB1+/+ controls were allowed to freely explore the EPM for 5 min. Merged heatmaps of all trials for (A) intCB1+/+ and (B) intCB1−/− mice show general exploration patterns of the open (vertical) and closed (horizontal) arms. Increasing time spent in area designated from blue to red, with red being most time. (C) IntCB1−/− female mice did not exhibit any differences in open-arm entries compared with controls [t(17)=1.588, p=0.1307]. (D) There were no differences in closed-arm entries between genotypes [t(16)=0.1938, p=0.8488]. (E) IntCB1−/− female mice and controls spent a similar amount of time exploring the open arms [t(17)=1.665, p=0.1142] and (F) closed arms of the EPM [t(17)=0.05312, p=0.9853]. There were no genotype differences in the (G) total number of head dips [t(17)=0.5512, p=0.5886], or the (H) cumulative time spent performing head dip behavior [t(17)=1.334, p=0.1999] in female mice. All analyses are unpaired Student's t-tests. Data presented as mean±SEM, n=9–10 mice per genotype.
FIG. 3.
FIG. 3.
Genotype differences in EPM exploration are not due to changes in movement. General movement parameters were quantified for both male and female mice on the EPM. (A) There were no differences in average velocity between male intCB1−/− mice and controls [t(20)=0.1997, p=0.8437] or (B) female intCB1−/− mice and controls [t(17)=1.394, p=0.1813]. (C) There were no differences in cumulative duration of movement between male intCB1−/− mice and controls [t(20)=0.7119, p=0.4847] or (D) female intCB1−/− mice and controls [t(17)=0.6774, p=0.5072]. (E) There were no differences in total distance traveled between male intCB1−/− mice and controls [t(20)=0.1986, p=0.8446] or (F) female intCB1−/− mice and controls [t(17)=1.427, p=0.1718]. (G) There were no differences in cumulative duration of nonmovement between male intCB1−/− mice and controls [t(20)=0.7119, p=0.4847] or (H) female intCB1−/− mice and controls [t(17)=0.8910, p=0.3854]. All analyses are unpaired Student's t-tests. Data presented as mean±SEM, n=9–11 mice per sex and genotype.
FIG. 4.
FIG. 4.
Male intCB1−/− mice, but not female, exhibit anxiolytic behaviors in the light/dark box. Male and female intCB1−/− mice and intCB1+/+ controls were allowed to freely explore the light/dark box for 10 min. (A) Merged heatmaps of all trials for male intCB1+/+, male intCB1−/− mice, female intCB1+/+, female intCB1−/− mice show general exploration patterns of the light box. Mice were unable to be recorded in the DB due to the opaque roof. Increasing time spent in area designated from blue to red, with red being most time. (B) Male intCB1−/− mice exhibited an increase in total light zone entries compared with controls, but there were no differences observed in light zone entries for female intCB1−/− mice and controls [sex×genotype interaction: F(1,37)=10.75; p=0.0023; sex main effect F(1,37)=6.236; p=0.0171; male intCB1−/− vs. male intCB1+/+ p=0.0053; two-way ANOVA followed by Holm-Sidak's multiple comparisons test]. (C) Male intCB1−/− mice exhibited an increase in light zone cumulative duration compared with controls, but there were no differences observed in light zone cumulative duration for female intCB1−/− mice and controls [sex×genotype interaction: F(1,36)=13.18; p=0.0009; sex main effect F(1,36)=22.21; p<0.0001; genotype main effect F(1,36)=4.521; p=0.0404; male intCB1−/− vs. male intCB1+/+ p=0.0008; two-way ANOVA followed by Holm-Sidak's multiple comparisons test]. Data presented as mean±SEM, n=9–12 mice per sex and genotype, **p<0.01, ***p<0.001. DB, dark box.
FIG. 5.
FIG. 5.
IntCB1−/− mice do not exhibit anxiolytic behaviors in the open field test. Male and female intCB1−/− mice and intCB1+/+ controls were allowed to freely explore the open field apparatus for 10 min. (A) Merged heatmaps of all trials for male intCB1+/+, male intCB1−/− mice, female intCB1+/+, female intCB1−/− mice show general exploration patterns of the open field. Increasing time spent in area designated from blue to red, with red being most time. There were no sex or genotype differences observed in (B) number of center zone entries, (C) cumulative duration in center zone, or (D) latency to first center zone entry. (E) IntCB1−/− female mice displayed a significant reduction in ambulation (total number of zones entered) compared with controls. There were no differences in ambulation between IntCB1−/− males and controls [genotype main effect F(1,37)=10.85; p=0.0022; female intCB1−/− vs. female intCB1+/+ p=0.0168]. (F) IntCB1−/− male and female mice demonstrated a reduction in total distance traveled compared with controls [genotype main effect F(1,37)=14.93; p=0.0004; male intCB1−/− vs. male intCB1+/+ p=0.0378; female intCB1−/− vs. female intCB1+/+ p=0.0037]. (G) IntCB1−/− male and female mice demonstrated a reduction in average velocity to controls [genotype main effect F(1,37)=14.96; p=0.0004; male intCB1−/− vs. male intCB1+/+ p=0.0379; female intCB1−/− vs. female intCB1+/+ p=0.0036]. (H) IntCB1−/− male and female mice demonstrated a reduction in the cumulative duration of movement to controls [genotype main effect F(1,38)=19.15; p<0.0001; male intCB1−/− vs. male intCB1+/+ p=0.0057; female intCB1−/− vs. female intCB1+/+ p=0.0057]. (I) IntCB1−/− male and female mice demonstrated an increase in the cumulative duration of movement compared with controls [genotype main effect F(1,37)=24.04; p<0.0001; male intCB1−/− vs. male intCB1+/+ p=0.0020; female intCB1−/− vs. female intCB1+/+ p=0.0020]. All analyses are two-way ANOVA followed by Holm-Sidak's multiple comparisons test. Data presented as mean±SEM, n=9–12 mice per sex and genotype. *p<0.05, **p<0.01.
FIG. 6.
FIG. 6.
Circulating CORT levels are sex dependent. Circulating CORT levels were quantified in male and female intCB1−/− mice and intCB1+/+ controls at baseline and after a 5-min EPM exposure. Correlation analysis was performed between post-EPM CORT levels and number of open-arm entries on the EPM per mouse. (A) There was a significant effect of timepoint and sex on plasma CORT levels. IntCB1+/+ female mice exhibited significantly higher CORT after EPM exposure when compared with intCB1+/+ female mice at baseline. IntCB1+/+ females post-EPM also exhibited a significant increase in CORT when compared with intCB1+/+ males post-EPM [timepoint main effect F(1,28)=24.44; p<0.0001; sex main effect F(1,28)=19.76; p=0.0001; **baseline female intCB1+/+ vs. post-EPM female intCB1+/+ p=0.0036; #post-EPM female intCB1+/+ vs. post-EPM male intCB1+/+ p=0.0311; three-way ANOVA followed by Holm-Sidak's multiple comparisons test]. (B) There were no significant differences in % change of plasma CORT. % Change=((Post-EPM CORT − Baseline CORT)/Baseline CORT)×100. Data presented as mean±SEM, n=7–9 mice group. ***p<0.001, ****p<0.0001. (C) Male intCB1+/+ mice exhibited an inverse correlation between post-EPM CORT levels and number of open-arm entries (p=0.02957, r2=0.6452), while intCB1−/− males did not (p=0.6631, r2=0.02869). (D) Female intCB1+/+ mice (p=0.1190, r2=0.3106) and intCB1−/− mice (p=0.05423, r2=0.5563) exhibited a nonsignificant trend toward an inverse correlation between post-EPM CORT levels and number of open-arm entries. CORT, corticosterone.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ruehle S, Rey AA, Remmers F, et al. . The endocannabinoid system in anxiety, fear memory and habituation. J Psychopharmacol 2012;26(1):23–39; doi: 10.1177/0269881111408958 - DOI - PMC - PubMed
    1. Petrie GN, Nastase AS, Aukema RJ, et al. . Endocannabinoids, cannabinoids and the regulation of anxiety. Neuropharmacology 2021;195:108626; doi: 10.1016/j.neuropharm.2021.108626 - DOI - PubMed
    1. Lutz B, Marsicano G, Maldonado R, et al. . The endocannabinoid system in guarding against fear, anxiety and stress. Nat Rev Neurosci 2015;16(12):705–718; doi: 10.1038/nrn4036 - DOI - PMC - PubMed
    1. Jenniches I, Ternes S, Albayram O, et al. . Anxiety, stress, and fear response in mice with reduced endocannabinoid levels. Biol Psychiatry 2016;79(10):858–868; doi: 10.1016/j.biopsych.2015.03.033 - DOI - PubMed
    1. Patel S, Hill MN, Cheer JF, et al. . The endocannabinoid system as a target for novel anxiolytic drugs. Neurosci Biobehav Rev 2017;76(Pt A):56–66; doi: 10.1016/j.neubiorev.2016.12.033 - DOI - PMC - PubMed

Publication types