Involvement of the accumbal osteopontin-interacting transmembrane protein 168 in methamphetamine-induced place preference and hyperlocomotion in mice

Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens

Overexpression of transmembrane protein 168 in the mouse nucleus accumbens induces anxiety and sensorimotor gating deficit

Transmembrane protein 168 (TMEM168) comprises 697 amino acid residues, including some putative transmembrane domains. It is reported that TMEM168 controls methamphetamine (METH) dependence in the nucleus accumbens (NAc) of mice. Moreover, a strong link between METH dependence-induced adaptive changes in the brain and mood disorders has been evaluated. In the present study, we investigated the effects of accumbal TMEM168 in a battery of behavioral paradigms. The adeno-associated virus (AAV) Tmem168 vector was injected into the NAc of C57BL/6J mice (NAc-TMEM mice). Subsequently, the accumbal TMEM168 mRNA was increased approximately by seven-fold when compared with the NAc-Mock mice (controls). The NAc-TMEM mice reported no change in the locomotor activity, cognitive ability, social interaction, and depression-like behaviors; however, TMEM168 overexpression enhanced anxiety in the elevated-plus maze and light/dark box test. The increased anxiety was reversed by pretreatment with the antianxiety drug diazepam (0.3 mg/kg i.p.). Moreover, the NAc-TMEM mice exhibited decreased prepulse inhibition (PPI) in the startle response test, and the induced schizophrenia-like behavior was reversed by pretreatment with the antipsychotic drug risperidone (0.01 mg/kg i.p.). Furthermore, accumbal TMEM168 overexpression decreased the basal levels of extracellular GABA in the NAc and the high K+ (100 mM)-stimulated GABA elevation; however, the total contents of GABA in the NAc remained unaffected. These results suggest that the TMEM168-regulated GABAergic neuronal system in the NAc might become a novel target while studying the etiology of anxiety and sensorimotor gating deficits

Increased anxiety and decreased PPI in the NAc-TMEM mice compared with the NAc-Mock mice.

<p>(A) Number of entries spent on open arms were measured for 10 min in the elevated plus-maze task; <i>N</i> = 9; Values are presented as mean ± S.E.M. *<i>p</i> < 0.05 vs. NAc-Mock (Student-<i>t</i> test). (B) Time spent on open arms was measured for 10 min in the elevated plus-maze task; <i>N</i> = 9; Values are presented as mean ± S.E.M. *<i>p</i> < 0.05 vs. NAc-Mock (Student-<i>t</i> test). (C) Time in the light box was measured for 10 min in the light/dark box task; N = 9; Values are presented as mean ± S.E.M. *<i>p</i> < 0.05 vs. NAc-Mock (Student-<i>t</i> test). (D) Startle responses were measured at 70, 80, 90, 100, 110, and 120 dB, respectively (background noise: 70 dB). <i>N</i> = 9; Values are presented as mean ± S.E.M. No significant difference between NAc-TMEM and NAc-Mock mice (ANOVA followed by the Bonferroni’s post hoc tests). (E) PPI was measured for 74, 78, 82, and 86 dB, respectively, of the prepulse intensity (background noise: 70 dB). Values are presented as mean ± S.E.M. <i>N</i> = 9. *<i>p</i> < 0.05 vs NAc-Mock (ANOVA followed by the Bonferroni’s post hoc tests).</p

Inhibitory effects of TMEM168 on GABA neurotransmission in the NAc.

<p>(A) Glutamate (GLU) and GABA concentrations in the NAc tissue were measured by HPLC. The NAc-Mock mice and NAc-TMEM mice were sacrificed and then the NAc tissue was extracted immediately, <i>N</i> = 11; values are presented as mean ± S.E.M. No significant difference between NAc-TMEM and NAc-Mock mice (two-way ANOVA followed by the Bonferroni’s post hoc tests). (B) Basal levels of extracellular GABA in the NAc were detected by the in vivo microdialysis task, <i>N</i> = 6; values are presented as mean ± S.E.M. *<i>p</i> < 0.05 vs. NAc-Mock (Student-<i>t</i> test). (C) Dynamic changes in the extracellular GABA levels in the NAc after high K⁺-stimulation was analyzed in the NAc by the in vivo microdialysis task, <i>N</i> = 6; values are given as mean ± S.E.M. **<i>p</i> < 0.01 vs. NAc-Mock. (ANOVA with repeated measures followed by the Bonferroni’s post hoc test) (D) Basal levels of extracellular dopamine in the NAc were detected by the in vivo microdialysis task, <i>N</i> = 4; values are presented as mean ± S.E.M. No significant difference between NAc-TMEM and NAc-Mock mice (Student-<i>t</i> test). (E) Basal levels of extracellular serotonin in the NAc were detected by the in vivo microdialysis task, <i>N</i> = 3–4; values are given as mean ± S.E.M. No significant difference between NAc-TMEM and NAc-Mock mice (Student-<i>t</i> test).</p

Reversal of sensorimotor gating deficit in the PPI task following the administration of risperidone in the NAc-TMEM mice.

<p>(A) Risperidone (0.01 mg/kg i.p.) or saline was administrated 30 min before the task performance. Startle responses was measured at 70, 80, 90, 100, 110, and 120 dB, respectively (background noise: 70 dB), <i>N</i> = 8; values are presented as mean ± S.E.M. No significant difference between NAc-TMEM and NAc-Mock mice (two-way ANOVA followed by the Bonferroni’s post hoc tests). (B) Risperidone (0.01 mg/kg i.p.) or saline was administered 30 min before the task performance. PPI was measured for 74, 78, 82, and 86 dB respectively, of prepulse intensity (background noise: 70 dB), <i>N</i> = 8; values are presented as mean ± S.E.M. **<i>p</i> < 0.01, *<i>p</i> < 0.05 vs. NAc-TMEM (VEH) (two-way ANOVA followed by the Bonferroni’s post hoc tests). VEH: saline administration group, RIS: risperidone administration group.</p

Involvement of the accumbal osteopontin-interacting transmembrane protein 168 in methamphetamine-induced place preference and hyperlocomotion in mice

Abstract Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens

Reversal of anxiety behaviors in the elevated plus-maze and light/dark box task following the administration of diazepam in the NAc-TMEM mice.

<p>(A) and (B) Diazepam (0.3 mg/kg i.p.) or vehicle was administered 30 min before performance in the elevated plus-maze task. Number of entries and time on open arms were measured for 10 min in the elevated plus-maze task, <i>N</i> = 6; Values are presented as mean ± S.E.M. *<i>p</i> < 0.05 vs. NAc-TMEM (VEH) (two-way ANOVA followed by the Bonferroni’s post hoc tests). (C) Diazepam (0.3 mg/kg i.p.) or vehicle was administered 30 min before the light/dark box test. Time in the light box was measured for 10 min in the light/dark box test, <i>N</i> = 8; values are presented as mean ± S.E.M. *<i>p</i> < 0.05 vs. NAc-TMEM (VEH) (two-way ANOVA followed by the Bonferroni’s post hoc tests); VEH: vehicle administration group, DZP: diazepam administration group.</p

Similar preferences in locomotor activity, Y-maze, novel object recognition, three chambers, tail suspension, and forced swimming tasks in the NAc-TMEM mice compared with the NAc-Mock mice.

<p>(A) The locomotor activity in the NAc-Mock and NAc-TMEM mice were measured for 60 min (No significant difference; Student-<i>t</i> test). (B) Working memory was assessed in the Y-maze spontaneous alternation task in the NAc-Mock and NAc-TMEM mice (No significant difference; Student-<i>t</i> test). (C) Cognitive function was assessed in the novel object recognition task. Percentage of total exploratory time on the novel object was expressed as exploratory preference (%) (No significant difference; ANOVA followed by the Bonferroni’s post hoc tests). (D) Social interaction was assessed in the three chambers task. Average time (10 min per phase) spent in the chamber with an object or a stranger mouse was detected (No significant difference; ANOVA followed by the Bonferroni’s post hoc tests). (E) Immobility time of the NAc–-Mock and NAc-TMEM mice in the tail-suspension task was measured for 5 min (No significant difference; Student-<i>t</i> test). (F) Immobility time of the NAc-Mock and NAc-TMEM mice in the forced swimming task was measured for 5 min (No significant difference; Student-<i>t</i> test). Values are presented as mean ± S.E.M. N = 9.</p