Transient receptor potential ankyrin 1 channel modulates the abuse-related mechanisms of methamphetamine through interaction with dopamine transporter

Abstract

Background and purpose: Methamphetamine (METH) use disorder has risen dramatically over the past decade, and there are currently no FDA-approved medications due, in part, to gaps in our understanding of the pharmacological mechanisms related to METH action in the brain.

Experimental approach: Here, we investigated whether transient receptor potential ankyrin 1 (TRPA1) mediates each of several METH abuse-related behaviours in rodents: self-administration, drug-primed reinstatement, acquisition of conditioned place preference, and hyperlocomotion. Additionally, METH-induced molecular (i.e., neurotransmitter and protein) changes in the brain were compared between wild-type and TRPA1 knock-out mice. Finally, the relationship between TRPA1 and the dopamine transporter was investigated through immunoprecipitation and dopamine reuptake assays.

Key results: TRPA1 antagonism blunted METH self-administration and drug-primed reinstatement of METH-seeking behaviour. Further, development of METH-induced conditioned place preference and hyperlocomotion were inhibited by TRPA1 antagonist treatment, effects that were not observed in TRPA1 knock-out mice. Similarly, molecular studies revealed METH-induced increases in dopamine levels and expression of dopamine system-related proteins in wild-type, but not in TRPA1 knock-out mice. Furthermore, pharmacological blockade of TRPA1 receptors reduced the interaction between TRPA1 and the dopamine transporter, thereby increasing dopamine reuptake activity by the transporter.

Conclusion and implications: This study demonstrates that TRPA1 is involved in the abuse-related behavioural effects of METH, potentially through its modulatory role in METH-induced activation of dopaminergic neurotransmission. Taken together, these data suggest that TRPA1 may be a novel therapeutic target for treating METH use disorder.

Keywords: addiction; dopamine transporter; methamphetamine; substance use disorder; transient receptor potential ankyrin 1 channel.

Figure 1.. TRPA1 antagonist treatment inhibited maintenance and reinstatement of METH IVSA.

Rats showing a stable METH IVSA (0.05 mg/kg/inj) were subjected to each experimental schedule. A967079 (10, 20, 40 mg/kg, i.p.) or vehicle was treated 30 min prior to the test sessions. (A) Experimental procedure of METH IVSA under fixed ratio schedule (n = 6 per group). (B) Number of injections, (C) Number of active lever presses, (D) Number of inactive lever presses. (E) Experimental procedure for METH IVSA under progressive ratio schedule (n = 8 per group), (F) Breakpoint, (G) Number of injections, (H) Number of active lever presses, (I) Number of inactive lever presses. (J) Experimental procedure for METH-primed reinstatement (n = 6 per group), (K) Number of active lever presses on the last day of each session, (L) Number of inactive lever presses on the last day of each session. Data were presented as mean ± SEM. Statistical analyses were performed by (B - D, K - L) repeated-measures ANOVA followed by Fisher’s LSD post hoc test or (F - I) paired t-test. Statistical significance was set at P < 0.05.

Figure 2.. TRPA1 mediates the development of METH-induced CPP and hyperlocomotion

(A) Experimental procedures (n = 10 per group). Mice were treated with either METH (1 mg/kg, i.p.) or saline during the conditioning sessions and on the day of open field test. (B) Effect of TRPA1 antagonist treatment on the METH-induced CPP. A967079 (10, 20, 40, 80 mg/kg, i.p.) or vehicle was treated 30 min prior to METH or saline administration. (C) METH-induced CPP in WT and TRPA1 KO mice. (D) Effect of TRPA1 antagonist treatment on the METH-induced hyperlocomotion. A967079 (10, 20, 40, 80 mg/kg, i.p.) or vehicle was treated 30 min prior to METH or saline administration. (E, F) METH-induced hyperlocomotion in WT and TRPA1 KO mice. Data were presented as mean ± SEM. Statistical analyses were performed by repeated-measures ANOVA followed by Tukey post hoc test. Statistical significance was set at P < 0.05.

Figure 3.. METH treatment increased dopamine levels only in WT mice but not in TPRA1 KO mice.

Test were performed on brain samples from mice treated with either saline or METH (1 mg/kg, i.p.) in CPP and open field test (n = 6 per group). Mice were sacrificed immediately after the open field test to obtain brain tissue samples. Dopamine levels in the (A) mPFC of WT mice, (B) NAc of WT mice, (C) VTA of WT mice, (D) mPFC of TRPA1 KO mice, (E) NAc of TRPA1 KO mice, (F) VTA of TRPA1 KO mice. Data were presented as mean ± SEM. Statistical analyses were performed by repeated-measures ANOVA followed by Tukey post hoc test. Statistical significance was set at P < 0.05.

Figure 4.. METH treatment increased the expression of dopamine-associated proteins only in WT mice but not in TPRA1 KO mice.

Test were performed on brain samples (NAc) from mice treated with either saline or METH (1 mg/kg, i.p.) in CPP and open field test (n = 7 per group). Mice were sacrificed immediately after the open field test to obtain brain tissue samples. Expression of (A) TRPA1-N in WT mice, (B) TRPA1-N in TRPA1 KO mice, (C) TRPA1-P in WT mice, (D) TRPA1-P in TRPA1 KO mice, (E) DAT in WT mice, (F) DAT in TRPA1 KO mice, (G) TH in WT mice, (H) TH in TRPA1 KO mice, (I) D1DR in WT mice, (J) D1DR in TRPA1 KO mice, (K) D2DR in WT mice, (L) D2DR in TRPA1 KO mice, (M) PKA in WT mice, (N) PKA in TRPA1 KO mice, (O) PKC in WT mice, (P) PKC in TRPA1 KO mice. Representative Western blot images are presented (top = target protein, bottom = β-actin). Protein expression levels were normalized relative to the β-actin, which served as the designated loading control. Data were presented as mean ± SEM. Statistical analyses were performed by repeated-measures ANOVA followed by Tukey post hoc test. Statistical significance was set at P < 0.05.

Figure 5.. TRPA1 mediates dopamine reuptake through interaction with DAT.

Cell lysates containing hDAT and TRPA1 were immunoprecipitated with FLAG beads (n = 3 per group). Dopamine reuptake assay was performed using HEK-293 cells stably expressing hDAT (n = 3 per group). Effect of TRPA1 agonist (AITC) or antagonist (A967079) treatment on the (A, D) Interaction between hDAT and TRPA1, (B, E) Dopamine reuptake activity of DAT, (C, F) Changes in dopamine reuptake activity of DAT induced by METH treatment. Data were presented as mean ± SEM. Statistical analyses were performed by (A, D) unpaired t-test or (B, C, E, F) repeated-measures ANOVA followed by Tukey post hoc test. Statistical significance was set at P < 0.05.

Figure 6.. Hypothetical diagram of the interaction between TRPA1 and DAT.

(A) In normal condition, dopamine is transported from the synaptic cleft into the presynaptic neuron via DAT. (B) Activation of TRPA1 enhances the interaction between TRPA1 and DAT, leading to the suppression of dopamine reuptake activity by DAT. (C) When TRPA1 is inactivated, the interaction between TRPA1 and DAT weakens, leading to an enhancement in the dopamine reuptake activity of DAT. Dopamine Transporter (DAT); Transient receptor potential channel Ankyrin 1 (TRPA1).