Loss of Sigma-2 Receptor/TMEM97 Is Associated with Neuropathic Injury-Induced Depression-Like Behaviors in Female Mice

Abstract

Previous studies have shown that ligands that bind to sigma-2 receptor/TMEM97 (s₂R/TMEM97), a transmembrane protein, have anxiolytic/antidepressant-like properties and relieve neuropathic pain-like effects in rodents. Despite medical interest in s₂R/TMEM97, little affective and pain behavioral characterization has been done using transgenic mice, which limits the development of s₂R/TMEM97 as a viable therapeutic target. Using wild-type (WT) and global Tmem97 knock-out (KO) mice, we sought to identify the contribution of Tmem97 in modulating affective and pain-like behaviors using a battery of affective and pain assays, including open field, light/dark preference, elevated plus maze, forced swim test, tail suspension test, and the mechanical sensitivity tests. Our results demonstrate that female Tmem97 KO mice show less anxiety-like and depressive-like behaviors in light/dark preference and tail suspension tests but not in an open field, elevated plus maze, and forced swim tests at baseline. We next performed spared nerve injury in WT and Tmem97 KO mice to assess the role of Tmem97 in neuropathic pain-induced anxiety and depression. WT mice, but not Tmem97 KO mice, developed a prolonged neuropathic pain-induced depressive-like phenotype when tested 10 weeks after nerve injury in females. Our results show that Tmem97 plays a role in modulating anxiety-like and depressive-like behaviors in naive animals with a significant change in the presence of nerve injury in female mice. Overall, these data demonstrate that Tmem97 could be a target to alleviate affective comorbidities of pain disorders.

Keywords: anxiety and depression; behaviors; pain; sigma-2 receptor/TMEM97.

Figure 1.

Visualization of Tmem97 expression in wild-type and global Tmem97 KO mice. Representative brain images of (A) WT and (E) global Tmem97 KO mice are labeled with in situ hybridization for Tmem97 (red) with DAPI (blue). Representative images for different regions of WT are shown including the (B) hippocampus, (C) dorsomedial hypothalamus, and (D) amygdala. Corresponding regions imaged at 40× with zoomed field of view are shown in the bottom panel: (B-40X) CA3 hippocampal layers, (C-40x) DMH, and (D-40X) amygdala subregions. DG, dentate gyrus; CeA, central amygdala; BLA, basolateral amygdala; DMH, dorsomedial hypothalamus; SR, stratum radiatum; SLu, stratum lucidum; SP, stratum pyramidal; SO, stratum oriens. Scale bar, 100 μm.

Figure 2.

Experimental timeline on baseline affective behaviors. Affective behaviors are performed on both WT and Tmem97 KO mice. Affective behaviors are performed in the order of open field, light/dark preference, elevated plus maze, elevated zero maze, forced swim test, and tail suspension test with 2 or 3 d in between the tests.

Figure 3.

Affective behaviors between wild-type and global Tmem97 KO naive mice. In naive mice, (A–C) there are no statistically significant differences in open-field behavior between WT and KO mice. B, Female mice show less anxiety compared with male mice irrespective of genotype. D–F, In the LDP test, female WT mice tend to show increased anxiety-like behaviors compared with female Tmem97 KO mice with a statistically significant difference observed in the (F) average time spent per visit. G, There was no trend for a difference in the LDP for latency to enter the light zone. H–J, In the EPM, there are no significant differences between WT and KO mice nor any obvious sex differences. Extended Data Figure 3-1 includes anxiety-like behaviors between WT and Tmem97 KO mice in both sexes using the EZM, another anxiety-like behavioral assay similar to EPM. K, In the FST, there are no differences between genotypes and sex in immobility behavior. L, In the TST, female Tmem97 KO mice show less depressive-like behaviors than female WT mice. OF, open field; LDP, light/dark preference; EPM, elevated plus maze; EZM, elevated zero maze; FST, forced swim test; TST, tail suspension test. Two-way ANOVA with Tukey's multiple comparison. Values are mean ± SEM, significant difference *p < 0.05, ****p < 0.0001.

Figure 4.

Analysis of test-specific and overall emotionality Z score to show combined effect of anxiety-like and depressive-like behaviors on wild-type and Tmem97 KO mice. The effect of combined anxiety-like and depressive-like behaviors across different parameters for each behavioral assay was calculated using Z score normalization. Lower value in Z score indicates less anxiety-like and depressive-like phenotypes. No significant difference was observed between WT and Tmem97 KO in Z score for (A) OF, (C) EPM, (D) EZM, and (E) FST. However, (B) in the LDP assay, female Tmem97 KO mice displayed a significant less Z score than wild-type mice. F, In the TST assay, female Tmem97 KO mice displayed a significantly less Z score than wild-type mice. G, The overall emotionality score shows that Tmem97 KO mice show less combine anxiety-like and depressive-like behavior compared with WT mice (main effect of genotype p = 0.0008). OF, open field; LDP, light/dark preference; EPM, elevated plus maze; EZM, elevated zero maze; FST, forced swim test; TST, tail suspension test. Two-way ANOVA with Tukey's multiple comparison. Values are mean ± SEM, significant difference *p < 0.05.

Figure 5.

Tmem97 is not associated with other behaviors related to executive function. A, Experimental timeline on behaviors testing executive functions is as follows. After handling for 2 consecutive days, behaviors consisting of nestlet shredding, marble burying assay, novel object recognition, and novel object location are performed on both WT and Tmem97 KO mice. No effect of sex or genotype was observed in (B) nestlet shredding, (C) marble burying, (D) novel object recognition, or (E) novel object location. NS, nestlet shredding, MB, marble burying, NOR, novel object recognition; NOL, novel object location. Relative discrimination index = [novel object (location) − familiar object (location)] / total exploration time of objects (location) (two-way ANOVA with Tukey's multiple-comparisons test). Values are mean ± SEM.

Figure 6.

Experimental timeline on prolonged neuropathic pain-induced affective behaviors. Affective behaviors (open field, light/dark preference, elevated plus maze, elevated zero maze, forced swim test, and tail suspension test) were performed on both WT and Tmem97 KO mice. Schematic of the SNI shows that the tibial and peroneal nerve are ligated and cut, leaving the sural nerve intact. Mechanical sensitivity is measured 2 d before the SNI and once every week (for 4 weeks) after the SNI was performed on a side of paw (in red) ipsilateral to injury. The affective behaviors are repeated >10 weeks after the SNI in the same manner as before the injury.

Figure 7.

Postoperative absolute change in affective behaviors from baseline to after the surgery stratified by sex and genotype. A–C, In OF, there were no significant differences between groups for absolute change from baseline. D–G, In LDP, there was a sex difference in absolute change from baseline within WT sham mice for (D) the number of light entries only. H, In the FST, female WT with SNI show significantly higher absolute changes from baseline compared with female sham WT mice consistent with a SNI-induced depressive-like effect in WT mice. I, In the TST, there is no statistically significant difference among groups. OF, open field; LDP, light/dark preference; FST, forced swim test; TST, tail suspension test. Three-way ANOVA allowing for different variances for genotype and adjusted for Bonferroni’s post hoc analysis. Values are mean ± SEM, significant difference *p < 0.05 and ***p < 0.001.

Figure 8.

Mechanical hypersensitivity before and after the SNI in wild-type and Tmem97 KO mice. No significant difference of mechanical hypersensitivity between WT and Tmem97 KO mice was observed at baseline and after SNI or sham surgery. Prolonged mechanical hypersensitivity was induced by SNI for WT and Tmem97 KO mice. Sham-treated group gradually recovered from acute mechanical hypersensitivity with full recovery by 4 weeks. Sham groups show statistically significant differences from the SNI group regardless of genotype. WT SNI (n = 13, 7 male and 6 female). WT sham (n = 11, 8 male and 3 female). Tmem97 KO SNI (n = 14, 7 male and 7 female). Tmem97 KO sham (n = 16, 8 male and 8 female). Extended Data Figure 8-1 shows a series of sensory-motor behavioral batteries performed with WT and Tmem97 KO mice to demonstrate that no motor deficits are observed, and affective behavioral phenotypes are independent of the surgery. BS, baseline. Repeated-measures, two-way ANOVA between group and time with Tukey's multiple-comparisons test within each time point. Values are mean ± SEM, no significant difference between the genotype within the same treatment. Significant difference between the treatment within the same genotype *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.