Article Figures & Data
Figures
- 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.
Tables
Figure Comparison Analysis p value, F, or t value N 3A Open field—total distance traveled by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.6430; F(1,68) = 0.22
Genotype: p = 0.8755; F(1,68) = 0.025
Interaction: p = 0.1968; F(1,68) = 1.70
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3B Open field—time spent in center zone by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: ****p < 0.0001; F(1,68) = 35.04
Genotype: p = 0.2163; F(1,68) = 1.56
Interaction: p = 0.0918; F(1,68) = 2.92
Sex effect
Male WT vs female WT: ****p < 0.0001
Male KO vs female KO: *p = 0.0160
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3C Open field—distance traveled in center zone by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.6736; F(1,68) = 0.18
Genotype: p = 0.8806; F(1,68) = 0.023
Interaction: p = 0.4798; F(1,68) = 0.50
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3D Light/dark preference—number of entries to light side by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.1888; F(1,68) = 1.76
Genotype: p = 0.0612; F(1,68) = 3.62
Interaction: p = 0.2503; F(1,68) = 1.34
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3E Light/dark preference—time spent in light side by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.0589; F(1,68) = 3.69
Genotype: *p = 0.0205; F(1,68) = 5.63
Interaction: p = 0.1534; F(1,68) = 2.08
Genotype effect
Male WT vs male KO: p = 0.9053
Female WT vs female KO: p = 0.0518
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3F Light/dark preference—average time spent per visit in light side by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: *p = 0.0137; F(1,68) = 6.40
Genotype: **p = 0.0097; F(1,68) = 7.08
Interaction: p = 0.1752; F(1,68) = 1.88
Sex effect
Male WT vs female WT: *p = 0.0487
Male KO vs female KO: p = 0.8262
Genotype effect
Male WT vs male KO: p = 0.7827
Female WT vs female KO: *p = 0.0358
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3G Light/dark preference—latency to first entry to light side by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.7564; F(1,68) = 0.097
Genotype: p = 0.5405; F(1,68) = 0.38
Interaction: p = 0.2777; F(1,68) = 1.20
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3H Elevated plus maze—total distance traveled by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: *p = 0.0421; F(1,51) = 4.35
Genotype: p = 0.2287; F(1,51) = 1.48
Interaction: p = 0.0773; F(1,51) = 3.25
Sex effect
Male WT vs female WT: p = 0.0635
Male KO vs female KO: p = 0.9964
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
3I Elevated plus maze—number of entries in open arm by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.4933; F(1,51) = 0.48
Genotype: p = 0.6114; F(1,51) = 0.26
Interaction: p = 0.1682; F(1,51) = 1.95
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
3J Elevated plus maze—time spent in open arm by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.8844; F(1,51) = 0.021
Genotype: p = 0.3175; F(1,51) = 1.02
Interaction: p = 0.6882; F(1,51) = 0.16
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
3K Forced swim test—duration of immobility by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.8739; F(1,68) = 0.025
Genotype: p = 0.3476; F(1,68) = 0.89
Interaction: p = 0.3526; F(1,68) = 0.88
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
3L Tail suspension test—duration of immobility by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.7254; F(1,68) = 0.12
Genotype: **p = 0.0013; F(1,68) = 11.24
Interaction: p = 0.1799; F(1,68) = 1.84
Genotype effect
Male WT vs male KO: p = 0.4683
Female WT vs female KO: *p = 0.0100
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
4A Z score on open field by genotype and sex Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.6953; F(1,68) = 0.15
Genotype: p = 0.6787; F(1,68) = 0.17
Interaction: p = 0.6480; F(1,68) = 0.21
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
4B Z score on light/dark preference Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.0509; F(1,68) = 3.95
Genotype: *p = 0.0106; F(1,68) = 6.90
Interaction: p = 0.0944; F(1,68) = 0.094
Genotype effect
Male WT vs male KO: p = 0.9045
Female WT vs female KO: *p = 0.0210
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
4C Z score on elevated plus maze Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.6683; F(1,51) = 0.19
Genotype: p = 0.4270; F(1,51) = 0.64
Interaction: p = 0.3565; F(1,51) = 0.87
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
4D Z score on elevated zero maze Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.1956; F(1,51) = 1.72
Genotype: p = 0.5834; F(1,51) = 0.30
Interaction: p = 0.5895; F(1,51) = 0.29
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
4E Z score on forced swim test Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.8752; F(1,68) = 0.025
Genotype: p = 0.3549; F(1,68) = 0.87
Interaction: p = 0.3454; F(1,68) = 0.9028
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
4F Z score on tail suspension test Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.7254; F(1,68) = 0.12
Genotype: **p = 0.0013; F(1,68) = 11.24
Interaction: p = 0.1799; F(1,68) = 1.84
Genotype effect
Male WT vs male KO: p = 0.4683
Female WT vs female KO: *p = 0.0100
Male WT = 18
Female WT = 15
Male Tmem97 KO = 20
Female Tmem97 KO = 19
4G Overall Z score on emotionality Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.9888; F(1,68) = 0.00020
Genotype: ***p = 0.0008; F(1,68) = 12.37
Interaction: p = 0.8085; F(1,68) = 0.059
Genotype effect
Male WT vs male KO: p = 0.0889
Female WT vs female KO: p = 0.0568
Male WT = 15–18
Female WT = 9–15
Male Tmem97 KO = 16–20
Female Tmem97 KO = 15–19
5B Nestlet shredding—percent mass change Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.6288; F(1,30) = 0.24
Genotype: p = 0.9455; F(1,30) = 0.0048
Interaction: p = 0.4405; F(1,30) = 0.61
Male WT = 11
Female WT = 5
Male Tmem97 KO = 12
Female Tmem97 KO = 6
5C Marble burying—number of marbles buried Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.0651; F(1,30) = 3.67
Genotype: p = 0.8778; F(1,30) = 0.024
Interaction: p = 0.1093; F(1,30) = 2.72
Male WT = 11
Female WT = 5
Male Tmem97 KO = 11
Female Tmem97 KO = 6
5D Novel object recognition for time investigated—relative discrimination index Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.9651; F(1,26) = 0.0019
Genotype: p = 0.5186; F(1,26) = 0.43
Interaction: p = 0.3282; F(1,26) = 0.99
Male WT = 11
Female WT = 5
Male Tmem97 KO = 8
Female Tmem97 KO = 6
5E Novel object location for number of entries investigated—relative discrimination index Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.4110; F(1,26) = 0.70
Genotype: p = 0.8019; F(1,26) = 0.064
Interaction: p = 0.6409; F(1,26) = 0.22
Male WT = 11
Female WT = 5
Male Tmem97 KO = 8
Female Tmem97 KO = 6
7A Open field—total distance traveled at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype Treatment: p = 0.9523; F(1,64) = 0.0036
Sex: p = 0.1051; F(1,64) = 2.7
Genotype: p = 0.3333; F(1,64) = 0.95
Treatment × sex: p = 0.2051; F(1,64) = 1.64
Treatment × genotype: p = 0.4616; F(1,64) = 0.55
Sex × genotype: p = 0.2563; F(1,64) = 1.31
Treatment × sex × genotype: p = 0.8964; F(1,64) = 0.017
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7B Open field—distance traveled in center at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype Treatment: p = 0.4413; F(1,64) = 0.60
Sex: p = 0.6033; F(1,64) = 0.27
Genotype: p = 0.1211; F(1,64) = 2.47
Treatment × sex: p = 0.2860; F(1,64) = 1.16
Treatment × genotype: p = 0.8159; F(1,64) = 0.055
Sex × genotype: p = 0.1890; F(1,64) = 1.76
Treatment × sex × genotype: p = 0.4828; F(1,64) = 0.50
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7C Open field—time spent in center at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype Treatment: p = 0.6740; F(1,64) = 0.18
Sex: p = 0.2267; F(1,64) = 1.49
Genotype: p = 0.2688; F(1,64) = 1.24
Treatment × sex: p = 0.5807; F(1,64) = 0.31
Treatment × genotype: p = 0.2760; F(1,64) = 1.21
Sex × genotype: p = 0.2691; F(1,64) = 1.24
Treatment × sex × genotype: p = 0.1349; F(1,64) = 2.29
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7D Light/dark preference—number of entries to light side at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype and adjusted for Bonferroni’s method (nonsignificant interaction effect dropped for post hoc purpose) Treatment: p = 0.1393; F(1,65.4) = 2.24
Sex: *p = 0.0235; F(1,62) = 5.40
Genotype: p = 0.1323; F(1,62.1) = 2.33
Sex × treatment: *p = 0.0300; F(1,65.4) = 4.92
Genotype × sex: *p = 0.0227; F(1,62.1) = 5.46
Sex × treatment effect: adjusted p value
Female SNI vs female sham: p = 0.9599
Female SNI vs male SNI: p = 0.9997
Female SNI vs male sham: *p = 0.0369
Female sham vs male SNI: p = 0.9350
Female sham vs male sham: *p = 0.0128
Male SNI vs male sham: *p = 0.0415
Genotype × sex effect: adjusted p value
KO female vs KO male: p = 1.000
KO female vs WT female: p = 0.0502
KO female vs WT male: p = 0.9394
KO male vs WT female: *p = 0.0478
KO male vs WT male: p = 0.9329
WT female vs WT male: *p = 0.0177
Significant post hoc sex effects
Sham WT male vs sham WT female: *p = 0.0011
SNI WT male vs SNI WT female: p = 0.3324
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7E Light/dark preference—time spent in light side at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype Treatment: p = 0.6966; F(1,64) = 0.15
Sex: p = 0.1308; F(1,64) = 2.34
Genotype: p = 0.4829; F(1,64) = 0.50
Treatment × sex: p = 0.3496; F(1,64) = 0.89
Treatment × genotype: p = 0.7381; F(1,64) = 0.11
Sex × genotype: p = 0.2320; F(1,64) = 1.46
Treatment × sex × genotype: p = 0.4074; F(1,64) = 0.70
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7F Light/dark preference—average time spent per visit in light side at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype Treatment: p = 0.6658; F(1,64) = 0.19
Sex: p = 0.8027; F(1,64) = 0.063
Genotype: p = 0.5007; F(1,64) = 0.46
Treatment × sex: p = 0.9823; F(1,64) = 0.00050
Treatment × genotype: p = 0.5690; F(1,64) = 0.33
Sex × genotype: p = 0.7305; F(1,64) = 0.12
Treatment × sex × genotype: p = 0.3753; F(1,64) = 0.80
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7G Light/dark preference—latency to first entry to light side at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype Treatment: p = 0.5038; F(1,64) = 0.45
Sex: p = 0.2468; F(1,64) = 1.37
Genotype: p = 0.5155; F(1,64) = 0.43
Treatment × sex: p = 0.3810; F(1,64) = 0.78
Treatment × genotype: p = 0.0935; F(1,64) = 2.90
Sex × genotype: p = 0.4062; F(1,64) = 0.70
Treatment × sex × genotype: p = 0.7113; F(1,64) = 0.14
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7H Time immobile in forced swim test at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype and adjusted for Bonferroni’s method (nonsignificant interaction effect dropped for post hoc purpose) Treatment: *p = 0.0221; F(1,66) = 5.50
Sex: p = 0.4848; F(1,66) = 0.49
Genotype: p = 0.1756; F(1,66) = 1.87
Sex × treatment: *p = 0.0440; F(1,66) = 4.21
Genotype × treatment: *p = 0.0155; F(1,66) = 6.17
Genotype × treatment effect: adjusted p value
KO SNI vs KO sham: p = 0.9997
KO SNI vs WT SNI: *p = 0.0365
KO SNI vs WT sham: p = 0.9078
KO sham vs WT SNI: *p = 0.0421
KO sham vs WT sham: p = 0.8642
WT SNI vs WT sham: **p = 0.0021
Sex × treatment effect: adjusted p value
Female SNI vs female sham: *p = 0.0186
Female SNI vs male SNI: p = 0.1972
Female SNI vs male sham: p = 0.1320
Female sham vs male SNI: p = 0.6580
Female sham vs Male sham: p = 0.7888
Male SNI vs male sham: p = 0.9953
Significant treatment post hoc effects
SNI WT female vs sham WT female: ***p = 0.0003
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
7I Time immobile in tail suspension test at pre- and postsurgery Three-way ANOVA with allowing different variances for genotype and adjusted for Bonferroni’s method (nonsignificant interaction effect dropped for post hoc purpose) Treatment: p = 0.6647; F(1,65.3) = 0.19
Sex: *p = 0.0282; F(1,60.6) = 5.06
Genotype: **p = 0.0066; F(1,60.6) = 7.91
Genotype × sex: *p = 0.0321; F(1,60.6) = 4.81
Genotype × sex effect: adjusted p value
KO female vs KO male: p = 1.0000
KO female vs WT female: **p = 0.0060
KO female vs WT male: p = 0.9763
KO male vs WT female: **p = 0.0048
KO male vs WT male: p = 0.9685
WT female vs WT male: *p = 0.0323
Significant sex post hoc effects
SNI WT male vs SNI WT female: p = 0.3273
Sham WT male vs sham WT female: p = 0.3798
Significant genotype post hoc effects
SNI WT female vs SNI KO female: p = 0.5202
Sham WT female vs sham KO female: p = 0.0652
Male WT SNI = 9
Male WT sham = 9
Male KO SNI = 10
Male KO sham = 10
Female WT SNI = 9
Female WT sham = 6
Female KO SNI = 9
Female KO sham = 10
8 Mechanical hypersensitivity across time Repeated-measures two-way ANOVA between time and group followed by Tukey's multiple-comparisons test at each time point Time: ****p < 0.0001; F(2.853,142.6) = 46.62
Group: ****p < 0.0001; F(3,50) = 16.62
Time × group: ****p < 0.0001; F(15,250) = 8.33
Baseline 1
WT SNI vs WT sham: p = 0.9594
WT SNI vs KO SNI: p = 0.9989
WT sham vs KO sham: p = 0.4629
KO SNI vs KO sham: p = 0.7256
Baseline 2
WT SNI vs WT sham: p = 0.9049
WT SNI vs KO SNI: p = 0.9661
WT sham vs KO sham: p = 0.2216
KO SNI vs KO sham: p = 0.9947
Post 7 d
WT SNI vs WT sham: *p = 0.0418
WT SNI vs KO SNI: p = 0.9263
WT sham vs KO sham: p = 0.9921
KO SNI vs KO sham: **p = 0.0015
Post 14 d
WT SNI vs WT sham: **p = 0.0022
WT SNI vs KO SNI: p = 0.7165
WT sham vs KO sham: p = 0.9520
KO SNI vs KO sham: ****p < 0.0001
Post 21 d
WT SNI vs WT sham: ****p < 0.0001
WT SNI vs KO SNI: p > 0.9999
WT sham vs KO sham: p = 0.8188
KO SNI vs KO sham: ***p = 0.0003
Post 28 d
WT SNI vs WT sham: ***p = 0.0002
WT SNI vs KO SNI: p = 0.9864
WT sham vs KO sham: p = 0.5849
KO SNI vs KO sham: ****p < 0.0001
WT sham = 11
WT SNI = 13
Tmem97 KO sham = 16
Tmem97 KO SNI = 14
3-1A Elevated zero maze—total distance traveled by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.2066; F(1,51) = 1.64
Genotype: p = 0.5897; F(1,51) = 0.29
Interaction: p = 0.1637; F(1,51) = 2.00
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
3-1B Elevated zero maze—number of entries in open arm by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.2613; F(1,51) = 1.29
Genotype: p = 0.6678; F(1,51) = 0.19
Interaction: p = 0.5986; F(1,51) = 0.28
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
3-1C Elevated zero maze—time spent in open arm by genotype Two-way ANOVA with Tukey's multiple-comparisons test Sex: p = 0.1635; F(1,51) = 2.00
Genotype: p = 0.5272; F(1,51) = 0.41
Interaction: p = 0.5967; F(1,51) = 0.28
Male WT = 15
Female WT = 9
Male Tmem97 KO = 16
Female Tmem97 KO = 15
8-1A Time taken for walk initiation test Repeated-measures mixed model ANOVA with allowing different variances for genotype and adjusted for Bonferroni’s method (nonsignificant interaction effect dropped for post hoc purpose) Time (pre/post): p = 0.2076; F(1,24) = 1.68
Genotype: p = 0.6009; F(1,23) = 0.28
Treatment: p = 0.2600; F(1,23) = 1.33
Time × treatment: *p = 0.0355; F(1,24) = 4.96
Time × treatment effect: adjusted p value
Pre SNI vs post SNI: p = 0.1041
Post sham vs post SNI: p = 0.2189
Pre sham vs post SNI: p = 0.3998
Pre SNI vs post sham: p = 0.9593
Pre sham vs pre SNI: p = 0.9996
Pre sham vs post sham: p = 0.9013
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1B Time taken to come down to the bottom of the pole Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): p = 0.1908; F(1,22) = 1.82
Genotype: p = 0.8413; F(1,22) = 0.04
Treatment: p = 0.8458; F(1,22) = 0.04
Time × genotype: p = 0.8469; F(1,22) = 0.04
Time × treatment: p = 0.1271; F(1,22) = 2.51
Genotype × treatment: p = 0.3423; F(1,22) = 0.94
Time × genotype × treatment: p = 0.2302; F(1,22) = 1.52
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1C Time taken to make the first turn on the pole Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): p = 0.5788; F(1,22) = 0.32
Genotype: p = 0.9752; F(1,22) = 0.00
Treatment: p = 0.4152; F(1,22) = 0.69
Time × genotype: p = 0.3913; F(1,22) = 0.76
Time × treatment: p = 0.4790; F(1,22) = 0.52
Genotype × treatment: p = 0.9425; F(1,22) = 0.01
Time × genotype × treatment: p = 0.9324; F(1,22) = 0.01
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1D Time stayed on the ledge without falling Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): p = 0.1869; F(1,22) = 1.86
Genotype: p = 0.1869; F(1,22) = 1.86
Treatment: p = 0.1869; F(1,22) = 1.86
Time × genotype: p = 0.1869; F(1,22) = 1.86
Time × treatment: p = 0.1869; F(1,22) = 1.86
Genotype × treatment: p = 0.1869; F(1,22) = 1.86
Time × genotype × treatment: p = 0.1869; F(1,22) = 1.86
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1E Time stayed on the platform without falling Repeated-measures Mixed Model ANOVA with allowing different variances for genotype Time (pre/post): p = 0.3829; F(1,22) = 0.79
Genotype: p = 0.3829; F(1,22) = 0.79
Treatment: p = 0.3829; F(1,22) = 0.79
Time × genotype: p = 0.3829; F(1,22) = 0.79
Time × treatment: p = 0.3829; F(1,22) = 0.79
Genotype × treatment: p = 0.3829; F(1,22) = 0.79
Time × genotype × treatment: p = 0.3829; F(1,22) = 0.79
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1F Latency time to go up to the top of the 60° inclined wire mesh Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): p = 0.4457; F(1,22) = 0.60
Genotype: p = 0.2325; F(1,22) = 1.51
Treatment: p = 0.6187; F(1,22) = 0.25
Time × genotype: p = 0.8701; F(1,22) = 0.027
Time × treatment: p = 0.5581; F(1,22) = 0.35
Genotype × treatment: p = 0.4541; F(1,22) = 0.58
Time × genotype × treatment: p = 0.3706; F(1,22) = 0.84
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1G Latency time to go down to the bottom of the 60° inclined wire mesh Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): ****p < 0.0001; F(1,22) = 33.51
Genotype: p = 0.1658; F(1,22) = 2.05
Treatment: p = 0.8178; F(1,22) = 0.05
Time × genotype: p = 0.1658; F(1,22) = 2.05
Time × treatment: p = 0.8178; F(1,22) = 0.05
Genotype × treatment: p = 0.7309; F(1,22) = 0.12
Time × genotype × treatment: p = 0.7309; F(1,22) = 0.12
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1H Latency time to go up to the top of the 90° inclined wire mesh Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): ***p = 0.0006; F(1,22) = 15.81
Genotype: p = 0.3877; F(1,22) = 0.78
Treatment: p = 0.4373; F(1,22) = 0.63
Time × genotype: p = 0.2603; F(1,22) = 1.34
Time × treatment: p = 0.1912; F(1,22) = 1.82
Genotype × treatment: p = 0.2726; F(1,22) = 1.27
Time × genotype × treatment: p = 0.4752; F(1,22) = 0.53
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1I Latency time to go down to the bottom of the 90° inclined wire mesh Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): **p = 0.0053; F(1,22) = 9.59
Genotype: p = 0.0704; F(1,22) = 3.62
Treatment: p = 0.4517; F(1,22) = 0.59
Time × genotype: p = 0.0704; F(1,22) = 3.62
Time × treatment: p = 0.4517; F(1,22) =0.59
Genotype × treatment: p = 0.6716; F(1,22) = 0.18
Time × genotype × treatment: p = 0.6716; F(1,22) = 0.18
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
8-1J Latency time to fall from inversed wire mesh Repeated-measures mixed model ANOVA with allowing different variances for genotype Time (pre/post): **p = 0.0086; F(1,22) = 8.32
Genotype: p = 0.6758; F(1,22) = 0.18
Treatment: p = 0.2138; F(1,22) = 1.64
Time × genotype: p = 0.3852; F(1,22) = 0.78
Time × treatment: p = 0.5172; F(1,22) = 0.43
Genotype × treatment: p = 0.9073; F(1,22) = 0.01
Time × genotype × treatment: p = 0.5435; F(1,22) = 0.38
WT sham = 7
WT SNI = 8
Tmem97 KO sham = 7
Tmem97 KO SNI = 4
Statistical significance was determined at the level of p < 0.05. Asterisks denoting p values include *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All data are presented as the mean ± standard error of the mean (SEM).
Figure 3-1
No significant difference between anxiety-like behaviors between wild-type and Tmem97 KO mice in both sexes using elevated zero maze. (A, B, C) In naïve mice, no significant difference between genotypes in both sexes is observed in any parameter of the EZM: total distance traveled, number of entries in open arm, and time spent in open arm. EZM = elevated zero maze. [Two-way ANOVA with Tukey's multiple comparison test]. Values are mean +/- SEM. Download Figure 3-1, TIF file.
Figure 8-1
Sensory-motor behavioral battery performed with wild-type and Tmem97 KO mice. No effect of genotype was observed in a battery of sensory-motor behaviors. No significant difference between genotypes is observed in (A) walk initiation, (B, C) turn on pole, (D) ledge time, or (E) platform time. (F - I) A trend for less latency to reach the bottom or top on a 60° and 90° inclined wire mesh was observed after surgery but there was no effect of genotype. (J) No significant difference was observed on the inverted wire mesh. [Repeated Measure, Mixed model ANOVA with allowing different variances for genotype and adjusted for Bonferroni method]. Values are mean +/- SEM, Significant difference. Download Figure 8-1, TIF file.
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