Effects of Acupuncture on Behavioral Stereotypies and Brain Dopamine System in Mice as a Model of Tourette Syndrome

Abstract

Tourette syndrome (TS), a developmental neurobehavioral disorder, is characterized by involuntary behavioral stereotypies. Clinical studies have confirmed the positive effect of acupuncture on treating TS, but the underlying mechanisms are not fully understood. In the present study, we used behavioral tests, Western blotting, double-immunofluorescence labeling, and fluorescence spectrophotometry to investigate whether acupuncture performed at acupoints "Baihui" (GV20) and "Yintang" (GV29) affected behavioral stereotypies and regulated the dopamine (DA) system in three different brain regions in Balb/c mice injected with 3,3'-iminodipropionitrile (IDPN) as a model for TS. We found that acupuncture alleviated behavioral stereotypies, down-regulated the expression of D1R and D2R in the striatum (STR) and substantia nigra pars compacta (SNpc), and decreased the concentration of DA in the STR, SNpc, and prefrontal cortex (PFC) as well. Moreover, acupuncture reduced the expression of tyrosine hydroxylase (TH) in the SNpc. Conclusively, acupuncture ameliorated behavioral stereotypies by regulating the DA system in the STR, SNpc, and PFC. Our findings provide novel evidence for the therapeutic effect of acupuncture on TS.

Keywords: Tourette syndrome; acupuncture; dopamine system; prefrontal cortex; striatum; substantia nigra pars compacta.

FIGURE 1

Effects of acupuncture on stereotyped behavior, motor coordination, and muscle strength in a mouse model of TS. (A) Evaluations of stereotyped behavior scores of mice in the control group (CON group), IDPN-induced TS model group (IDPN group), IDPN-induced TS model with acupuncture with twisting group (ACU group), IDPN-induced TS model with acupuncture without twisting group (SHAM1 group), and IDPN-induced TS model with acupuncture on non-acupoints with twisting group (SHAM2 group) on days 0, 8, 15, 22, 29, and 36. (B) Latency to fall in the rotarod test among the CON, IDPN, ACU, SHAM1, and SHAM2 groups on days 0, 8, 15, 22, 29, and 36. (C) Forelimb grip force among the CON, IDPN, ACU, SHAM1, and SHAM2 groups on days 0, 8, 15, 22, 29, and 36. Data are expressed as means ± SEM (n = 18 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group.

FIGURE 2

Effects of acupuncture on DA concentration in the STR, SNpc, and PFC. (A) Quantitative analysis of DA concentration in the STR of different groups. (B) Quantitative analysis of DA concentration in the SNpc of different groups. (C) Quantitative analysis of DA concentration in the PFC of different groups. (D) Quantitative analysis of DA concentration in the thalamus of different groups. Data are expressed as means ± SEM (n = 6 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group; ⁺P < 0.05, compared with the ACU group.

FIGURE 3

Effects of acupuncture on TH protein expression and the number of TH positive neurons in the SNpc. (A) Representative gel images showed the protein level of TH in the SNpc tissues obtained from the CON, IDPN, ACU, SHAM1, and SHAM2 groups. GAPDH was used as a loading control. (B) TH labeling (green); NeuN labeling (red); DAPI (blue). Scale bar, 100 μm. (C) Summary data showed effects of IDPN and acupuncture on the protein level of TH in the SNpc. (D) Summary graph shows the percentage of double-labeled TH and NeuN immunoreactivity in the total of NeuN-positive cells in the SNpc from different groups. Data are expressed as means ± SEM (n = 6 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group; ⁺P < 0.05, compared with the ACU group.

FIGURE 4

Effects of acupuncture on D1R and D2R protein expression in the STR. (A) Representative gel images showed the protein levels of D1R and D2R in the STR tissues obtained from the CON, IDPN, ACU, SHAM1, and SHAM2 groups. GAPDH was used as a loading control. (B) Summary data showed the effects of IDPN and acupuncture on protein level of D1R in the STR. (C) Summary data showed the effects of IDPN and acupuncture on protein level of D2R in the STR. Data are expressed as means ± SEM (n = 6 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group; ⁺P < 0.05, compared with the ACU group.

FIGURE 5

Effects of acupuncture on D1R and D2R protein expression in the SNpc. (A) Representative gel images showed the protein levels of D1R and D2R in the SNpc tissues obtained from the CON, IDPN, ACU, SHAM1, and SHAM2 groups. GAPDH was used as a loading control. (B) Summary data showed the effects of IDPN and acupuncture on protein level of D1R in the SNpc. (C) Summary data showed the effects of IDPN and acupuncture on protein level of D2R in the SNpc. Data are expressed as means ± SEM (n = 6 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group; ⁺P < 0.05, compared with the ACU group.

FIGURE 6

Effects of acupuncture on the number of D1R and D2R positive neurons in the STR. (A) D1R labeling (green); NeuN labeling (red); DAPI (blue). Scale bar, 100 μm. (B) Summary graph shows the percentage of double-labeled D1R and NeuN immunoreactivity in the total of NeuN-positive cells in the STR from different groups. (C) D2R labeling (green); NeuN labeling (red); DAPI (blue). Scale bar, 100 μm. (D) Summary graph shows the percentage of double-labeled D2R and NeuN immunoreactivity in the total of NeuN-positive cells in the STR from different groups. Data are expressed as means ± SEM (n = 6 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group; ⁺P < 0.05, compared with the ACU group.

FIGURE 7

Effects of acupuncture on the number of D1R and D2R positive neurons in the SNpc. (A) D1R labeling (green); NeuN labeling (red); DAPI (blue). Scale bar, 100 μm. (B) Summary graph shows the percentage of double-labeled D1R and NeuN immunoreactivity in the total of NeuN-positive cells in the SNpc from different groups. (C) D2R labeling (green); NeuN labeling (red); DAPI (blue). Scale bar, 100 μm. (D) Summary graph shows the percentage of double-labeled D2R and NeuN immunoreactivity in the total of NeuN-positive cells in the SNpc from different groups. Data are expressed as means ± SEM (n = 6 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group; ⁺P < 0.05, compared with the ACU group.

FIGURE 8

Effects of acupuncture on D1R protein expression and the number of D1R-positive neurons in the PFC. (A) Representative gel images showed the protein levels of D1R in the PFC tissues obtained from the CON, IDPN, ACU, SHAM1, and SHAM2 groups. GAPDH was used as a loading control. (B) D1R labeling (green); NeuN labeling (red); DAPI (blue). Scale bar, 100 μm. (C) Summary data showed the effects of IDPN and acupuncture on the protein level of D1R in the PFC. (D) Summary graph show the percentage of double-labeled D1R and NeuN immunoreactivity in the total of NeuN-positive cells in the PFC from different groups. Data are expressed as means ± SEM (n = 6 mice in each group). ^∗P < 0.05, compared with the CON group; ^#P < 0.05, compared with the IDPN group; ⁺P < 0.05, compared with the ACU group.