Long-term consequences of adolescent exposure to the synthetic cannabinoid AB-FUBINACA in male and female mice

Abstract

The consumption of synthetic cannabinoids during adolescence is reported to be a risk factor for the appearance of psychiatric disorders later in life. AB-FUBINACA is a member of the indazole carboxamide family of synthetic cannabinoids present in Spice/K2 preparations. The present study sought to investigate the long-term effects of AB-FUBINACA consumption during adolescence in both male and female mice. AB-FUBINACA revealed several sex-dependent behavioral alterations. In this sense, the administration of this synthetic cannabinoid in female, but not male, mice induced psychotic-like symptoms which were associated with changes in dendritic arborization and density of mature dendritic spines in pyramidal neurons of the prefrontal cortex, as well as with an up-regulation of differentially expressed genes in this brain area. This study helps to clarify the potential late detrimental effects of this potent synthetic cannabinoid that may derive from its use during adolescence.

Keywords: behavioral neuroscience; biological sciences; natural sciences; neuroscience.

Graphical abstract

Figure 1

AB-FUBINACA treatment during adolescence alters anxiety-like behavior in a sex-dependent manner (A) Schematic representation of experimental design. (B–E) Effects of adolescent exposure to AB-FUBINACA (PND 35–39: 1 mg/kg, PND 40–44: 1.5 mg/kg, and PND 45–49: 2 mg/kg) or vehicle in anxiety-like behavior in the EPM (B, C) and fear extinction (D, E) in male (B, D) and female (C, E) mice (n = 12 mice per group). Percentage of time spent in the open arm and total number of entries are shown for the EPM. Time course of the freezing levels scored during cued fear extinction trials is shown for fear memory processing. Data are expressed as mean ± SEM. ∗p < 0.05 (comparison between AB-FUBINACA and vehicle; Student’s t test). PND postnatal day, EPM elevated plus maze, LOC locomotion, E1-E5 extinction trials.

Figure 2

AB-FUBINACA treatment during adolescence alters novel object recognition memory and depressive-like behavior in a sex dependent manner (A) Schematic representation of experimental design. (B–G) Effects of adolescent exposure to AB-FUBINACA (PND 35–39: 1 mg/kg, PND 40–44: 1.5 mg/kg, and PND 45–49: 2 mg/kg) or vehicle in memory in the NOR (B, E), sociability in the three chamber test (C, F), and depressive-like behavior in the FST (D, G) in male (B, C, D) and female (E, F, G) mice (n = 11–15 mice per group). The discrimination index is shown for the NOR, total time in direct contact with each compartment is shown for sociability test, and immobility time is shown in the FST. Data are expressed as mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001 (comparison between AB-FUBINACA and vehicle group; two-way ANOVA, compartment (C, F); Student’s t test (D, E). PND postnatal day, NOR novel object recognition, FST forced swimming test.

Figure 3

AB-FUBINACA treatment during adolescence alters sensorimotor gating in a sex-dependent manner (A) Schematic representation of experimental design. (B) Graphic diagram of the PPI protocol. (C–H) Effects of adolescent exposure to AB-FUBINACA (PND 35–39: 1 mg/kg, PND 40–44: 1.5 mg/kg, and PND 45–49: 2 mg/kg) or vehicle in sensorimotor gating in male (C, D, E) and female (F, G, H) mice (n = 13–15 mice per group). Percentage of prepulse inhibition (C, F), mean of the percentage of prepulse inhibition (D, G), and startle response amplitude (E, H) are shown. Data are expressed as mean ± SEM. ∗p < 0.05 (comparison between AB-FUBINACA and vehicle group; two-way ANOVA with repeated measures, treatment (F); Student’s t test (G). PND postnatal day, PPI prepulse inhibition test, dB decibels, ITI inter-trial interval.

Figure 4

AB-FUBINACA treatment during adulthood alters sensorimotor gating, but neither anxiety-like behavior nor memory, in female mice (A) Schematic representation of experimental design. (B–F) Effects of adult exposure to AB-FUBINACA (PND 69–73: 1 mg/kg, PND 74–78: 1.5 mg/kg, and PND 79–83: 2 mg/kg) or vehicle in anxiety-like behavior in the EPM (B), memory in the NOR (C), and sensorimotor gating (D, E, F) in female mice (n = 10–17 mice per group). Percentage of time spent in the open arm and total number of entries are shown for the EPM, the discrimination index and total time of exploration are represented for the NOR. Percentage of prepulse inhibition (D), mean of the percentage of prepulse inhibition (E), and startle response amplitude (F) are also shown. Data are expressed as mean ± SEM. ∗p < 0.05 (comparison between AB-FUBINACA and vehicle group; two-way ANOVA with repeated measures, treatment (D); Student’s t test (E). PND postnatal day, EPM elevated plus maze, NOR novel object recognition, PPI prepulse inhibition test, dB decibels.

Figure 5

AB-FUBINACA treatment during adolescence induces an up-regulation of PLEKHG2 and SH3TC1 genes in the prefrontal cortex of adult females (A) Schematic representation of experimental design. (B) Volcano plot summarizing DEGs of AB-FUBINACA vs. vehicle treated mice (n = 4 mice per group). (C) DEGs in each AB-FUBINACA and vehicle treated mouse clustered with a heatmap. (D and E) Correlation between percentage of prepulse inhibition and Plekhg2 (D) and Sh3tc1 (E) relative expression (Pearson correlation coefficient). PND postnatal day, PPI prepulse inhibition test, PFC prefrontal cortex, DEGs differentially expressed genes.

Figure 6

AB-FUBINACA treatment during adolescence induces alterations in dendritic arborization and dendritic spine density in the prefrontal cortex of adult females Effects of treatment with AB-FUBINCA or vehicle during adolescence in dendritic arborization (A-E) and dendritic spine density (F-I) in the prefrontal cortex of adult female mice. (A) Representative traces of reconstructed neurons in AB-FUBINACA or vehicle-treated female mice. Scale bar = 50 μm. (B) Sholl analysis represented by the number of intersections every 20 μm. (C) Total dendritic length. (D) Volumetric data of neuron dendrites in μm3. (E) Plot comparing the total number of reconstructed neurons (n = 6 neurons/mouse and n = 4 mice per group) across principal components PC1 and PC2. (F) Representative images of apical dendritic spines from AB-FUBINACA and vehicle treated female mice. Scale bar = 2 μm. (G) Total spine density of apical dendrites in AB-FUBINACA and vehicle treated female mice (n = 4–5 neurons/mouse and n = 4 mice per group). (H) Spine density grouped according to their morphological characteristics in apical dendrites. (I) Schematic representation of the morphological classification of the dendritic spines. Data are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 (comparison between AB-FUBINACA and vehicle group; mixed-model ANOVA, interaction treatment x radius (B); Mann-Whitney U test (C); Student’s t test (D, G, H). PC principal components 1 and 2.