Genetic markers of a Munc13 protein family member, BAIAP3, are gender specifically associated with anxiety and benzodiazepine abuse in mice and humans

Abstract

Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders.

Figure 1

Immunofluorescence analysis of Baiap3 expression in mouse brain. (A) Sagittal brain section of adult Baiap3 WT mouse stained with rabbit anti-Baiap3 antibody. (B) Sagittal brain section of adult Baiap3 KO littermate showing the absence of Baiap3 immunoreactivity. Please note that the signal observed in the hippocampus of both WT and KO brain is a nonspecific background staining. (C) Coronal brain section of adult WT mouse stained for Baiap3 with a corresponding coronal diagram, adapted from the mouse Paxinos brain atlas (Bregma −1.46). PB, parabrachial nucleus; NTS, nucleus tractus solitarius; Hi, hippocampus; SC, superior colliculus; IC, inferior colliculus; PAG, periaqueductal gray; LS, lateral septum; Th, thalamus; Hy, hypothalamus; BST, bed nucleus of the stria terminalis; PV, paraventricular thalamic nucleus; DM, dorsomedial hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; Arc, arcuate nucleus; Ce, central amygdaloid nucleus; BLA, basolateral amygdaloid nucleus, anterior part; BMA, basomedial amygdaloid nucleus, anterior part; ME, medial amygdaloid nucleus; ACo, anterior cortical amygdaloid nucleus. Scale bars equal 1 mm.

Figure 2

Anxiety phenotype in Baiap3 KO mice. (A–F) Open field parameters. (A, B) The latency to reach the wall of the open field was significantly increased in Baiap3 KO mice of both sexes, whereas visits to the center (C, D) and stay in the periphery (E, F) revealed anxiety-like behavior only in females. Elevated plus-maze (G, H) and light–dark box (I, J) revealed no genotype-dependent differences in either sex. (K, L) As readout of unspecific novelty-related anxiety, a higher freezing response was found in male as well as female Baiap3 KO mice. Numbers tested: males, WT = 16–25, KO = 16–25; females, WT = 18–23, KO = 10–28. Mann-Whitney U test (A–D, I–L) and two-way ANOVA (E–H), including Bonferroni testing, were applied. Means ± SEM are presented.

Figure 3

Diazepam tolerance and withdrawal in Baiap3 KO and WT mice. (A) Experimental design scheme. (B) Male diazepam-treated Baiap3 KO mice showed significantly faster improvement of performance on the rotarod, consistent with a more rapid development of tolerance to diazepam. (C) Rotarod performance of female mice was comparable between WT and KO. (D, E) Diazepam-naive Baiap3 KO mice display a higher PTZ-induced seizure propensity compared with WT (significant in females, strong tendency in males). (F, G) Flumazenil-induced diazepam withdrawal does not further increase PTZ-induced seizure propensity in Baiap3 KO mice. Seizure propensity of female mice became comparable between genotypes, pointing to a ceiling effect. Numbers tested: males, WT = 25, KO = 25; females, WT = 21, KO = 23, except for (D) and (E), where males, WT = 7, KO = 7; females, WT = 8; KO = 10. Mann-Whitney U test (D–G) and two-way repeated-measures ANOVA (B, C), including Bonferroni, testing applied. Means ± SEM are presented.

Figure 4

Increased basal network activity and lack of homeostatic adaptation to diazepam treatment in Baiap3 KO hypothalamus slices. (A) Sample traces of mIPSC recordings from WT and KO hypothalamus slices that were cultured in the presence of diazepam or under vehicle control conditions with DMSO. Under diazepam withdrawal conditions, Baiap3 WT mIPSC amplitudes were significantly smaller than in KO slices (B), and WT mIPSC rise times were longer than in KO slices (C). (D) Baiap3 WT slices showed an increase in AP frequency in response to diazepam withdrawal when compared with DMSO-treated WT slices, whereas no such increase was apparent for Baiap3 KO slices, which already showed an increased AP frequency under DMSO control conditions when compared with WT slices. (E) The resting membrane potential was not affected by experimental condition or Baiap3 genotype. IPSC amplitudes (F) and IPSC frequencies (G) were increased in Baiap3 WT slices under diazepam withdrawal compared with DMSO-treated WT slices. Mann-Whitney U test was used for AP and IPSC frequencies; Student t test was used for all other parameters. Means ± SEM are presented.