Acute MPTP administration modeling process was showed in Fig. 1(a): two monkeys survived after acute MPTP administration (0.5 mg/kg, 7 daily injections). Tremor frequency and walking time were used to evaluate the treatment effect. Fig. 1(b) showed the tremor frequency of the two advanced PD monkeys temporarily decreased significantly after M1 tDCS (0.3 mA, 10 min). Amazingly, these two advanced PD monkeys could not move before M1 tDCS, but they walked for a while immediately after M1 tDCS (Fig. 1(c)) and they could sit in the cage instead of lying down as before tDCS treatment. These data indicated that M1 tDCS temporarily improved PD monkeys’ motor symptoms dramatically. Unluckily, these two monkeys died after three days, indicating M1 tDCS could not cure PD thoroughly although its temporary treatment effects were excellent.

Figure 1 The temporary effects of anodal tDCS on M1 in two advanced PD monkeys (1#, 2#). (a) Acute modeling process of MPTP administration. (b) Tremor frequency of PD monkeys was dramatically improved by anodal tDCS on M1. (One-way ANOVA, Monkey 1#: **P = 0.0002; Independent Sample Mann-Whitney U Test, Monkey 2#: **P = 3.3 × 10−9). (c) Voluntary locomotion of PD monkeys was obviously increased after anodal tDCS on M1. Values in (b,c) represent mean ±STD. Full size image

To detect the long-term treatment effects of tDCS and find the main parameters of anodal tDCS on M1 with intensity from 0.3 (referred to the monkeys 1# and 2#), 0.4 (higher) to 0.2 (lower) mA and time from 5, 10, 15 to 20 minutes, another two monkeys (3# and 4#) were used in this experiment (Fig. 2(a)). The PD scores before tDCS showed a mild decrease with stimuli trails, but they were not larger than 2, which meant little medical significance. The PD score of Kurlan scale was dramatically decreased right after anodal tDCS, but was restored to the pretherapeutic level before the next tDCS treatment (Fig. 2(b)). This fast recovery indicated the temporarily therapeutic effects of a single tDCS treatment which disappeared in one day.

Figure 2 The accumulated effects of anodal tDCS on M1 in two advanced monkeys (3# and 4#). (a) Mild-acute modeling process of MPTP administration. (b) Consecutive treatment of anodal tDCS on M1. The intensity of tDCS ranges from 0.2 to 0.4 mA and time from 5 to 20 min. (The treatment effect is statistically significant before and after tDCS: Related-Samples Wilcoxon Signed Rank Test for Monkey 3#: **P = 0.002, Paired t-test for Monkey 4#: **P < 0.01). (c) The quantitative relationship between accumulated stimulation (∑ intensity × duration) and anodal tDCS treatment effects (PD Score before tDCS − PD Score after tDCS ) on M1 monkeys 3# and 4#. Grey squares represent the average tDCS treatment effects at different accumulated stimulation of each monkey. Values in (b,c) represent mean ± STD. Full size image

However, the changes of PD score after tDCS were significantly decreased over 7 after the consecutive 12-days treatments. Multivariate linear regression was used to evaluate the effects of parameters (monkey, stimuli duration, stimuli intensity, accumulated stimulation) on the decrease. The result showed that the accumulated stimulation (P < 0.01) instead of monkey (P = 0.076), stimuli intensity (P = 0.065) or stimuli duration (P = 0.058) could explain the changes. These results suggested that although single tDCS treatment could not cure PD thoroughly, consecutive treatments of tDCS with accumulated effects were very effective to improve tDCS effect. The more accumulated stimulation of tDCS, the better treatment effect. The relationship between treatment effects (PD Score before tDCS − PD Score after tDCS ) and accumulated stimulation of tDCS (∑intensity × time) was quantitatively described in Fig. 2(c), which showed a linear increase.

Based on the accumulated treatment effects of tDCS, we wanted to know whether the accumulated effect would last at least for six months. After six months’ natural recovery, the PD scores of two monkeys were declined to 5 and 9 respectively. Therefore, the two monkeys received additional MPTP administration (0.45 mg/kg, 5 daily injections) to make sure their PD scores were increased to 16–18 again (Fig. 3(a)). Then the two monkeys were subjected to a single tDCS treatment (0.2 mA, 5 min). The result showed that the therapeutic effect of the single tDCS treatment was not different from the effect of six months before (first tDCS treatment in Fig. 2(b)) (Fig. 3(b)), indicating an extinction of accumulated effect of tDCS six months after. Furthermore, the recovery of PD score in each monkey may be attributed to the compensatory mechanism24,25,26 but not the accumulated effects of anodal tDCS on M1, since the accumulated effect of tDCS treatments was disappeared during the six-month period.

Figure 3 Extinction of tDCS accumulated treatment effects and contributions of different brain areas to tDCS effects for PD treatment. (a) After six months’ recovery, monkey 3# and 4# were administered with additional MPTP to restore the Kurlan scale scores same to the scores in Fig. 2(a). (b) Extinction of tDCS accumulated treatment effects on M1 after six months’ recovery. There was no significant difference between the effects of the first tDCS and the last tDCS in each monkey (Multi-factor ANOVA: P = 0.881). (c) The effects of tDCS treatment (0.2 mA, 5 min) on different brain areas. The PD scores only decreased after tDCS on M1. Sham: control of anodal tDCS on M1 with no stimulation; PFC: prefrontal cortex; LT: left temporal lobe; RT: right temporal lobe; M1: primary motor cortex. Multi-factor ANOVA: The interaction of region × tDCS was significant, **P < 0.01. Values in (b,c) represent mean ± STD. Full size image

In addition to M1, we also tested other brain areas to ascertain whether these areas contribute to tDCS treatment effects. The results showed that there were no significant difference in treatment effects before and after tDCS treatment on prefrontal cortex (PFC), left temporal lobe (LT) or right temporal lobe (RT) (Fig. 3(c)). These data indicated that stimulation on M1 rather than other brain areas could induce a significant treatment effect of PD motor symptoms.

Importantly, what is the neuronal mechanism underlying the anodal tDCS on M1? To answer this question, we checked the neuronal activation by c-fos staining after anodal tDCS on M1. Unfortunately, the monkey 3# was died after being finished the experiments above, so we only used monkey 4# for c-fos staining. The most effective and economic design for this study is the self-control in monkey 4#: anodal tDCS on right side of M1 and the left side as the baseline of c-fos expression. Before this experiment, we confirmed the tDCS treatment effect again (the behavior analysis and video clips were provide in Supplementary Data). To avoid unnecessary stimulations activating the c-fos expression, this experiment was done in a silent and dark room. After anodal tDCS on right side of M1 (0.3 mA, 10 min, 3 times repeated), the monkey 4# stayed in the room for 2 hours for the time of c-fos expression and then was sacrificed for c-fos immunohistochemical examinations. The results showed that tDCS on M1 significantly activated the c-fos expression in the neurons of M1 and SN compared to the basal level (Fig. 4). It is indicated that anodal tDCS on M1 may active the neurons in M1 and SN in the neural circuits associated with motor control, which would improve the PD behaviors.

Figure 4 C-fos staining of the neurons in M1 and SN after anodal tDCS on the right side of M1. (a) The c-fos immunohistochemical staining of left and right side neurons in M1. C-fos positive cells were counted for analysis. (b) The c-fos expression in the neurons of the right side M1 was significantly increased compared to the basal level. Paired t-test: **P = 0.0007. (c) The c-fos staining of left and right side neurons in SN. (d) The c-fos expression in the neurons of the right side SN was significantly increased. Paired t-test: ##P = 0.0009. Values in (b,d) represent mean ±STD. Scale bar: 20 um. Full size image

Finally, we verified this advanced PD monkey model by TH immunohistochemical staining. The results showed that TH positive neurons in the SNc were significantly lost (more than 80%) in MPTP treated monkeys compared to normal controls and the morphology of neurons in MPTP treated groups was pathologically changed (Fig. 5). This data was accorded to the advanced PD patients, indicating this model used here was reliable and the tDCS treatment effects were also potentially to be applied in clinic.