Behavioural Testing

METH-induced sensitization was performed as previously described (Fig. 1A) [25]. Briefly, METH-sensitized group received repeated doses of 1 mg/kg METH on days 1 and 7; 5 mg/kg, i.p. on days 2–6. The acute METH and saline groups were injected with saline for 7 days. Then, after a 2-week withdrawal period, the METH-sensitized and acute METH group received the same challenge dose of METH (1 mg/kg), while the saline group received one injection of saline on day 23 (Fig. 1A). Horizontal locomotor activities were recorded in metal test chambers (43 cm × 43 cm × 43 cm) and analysed for 60 min after 1 mg/kg METH injections using a smart 2.5 video tracking system. The amount of time travelled in the centre zone (21.50 cm × 21.50 cm) are interpreted as measures of anxiety-like behaviour [36].

A–E Changes in Notch1 signalling in the mPFC of METH-sensitized mice. A Procedure for generating the METH-induced locomotor sensitization model. B Representative mouse tracks from days 1, 7 and 23 were illustrated. C METH-induced locomotor sensitization in mice. One-way repeated-measures ANOVA revealed a significant main effect of METH [F _{(2, 21)} = 47.41, P < 0.001]. Subsequent post hoc LSD comparisons found significantly greater locomotor activity in the METH-sensitized group than in the saline group on day 1, day 7 and day 23. The METH-sensitized group showed a significant increase in locomotor activity compared with the METH-acute group on day 23 and itself on day 1. D The amount of time mice spent in the centre zone significantly decreased in the METH-sensitized group compared with the saline and acute METH group on day 23 [One-way repeated-measures ANOVA, F _{(2, 15)} = 3.66_, P < 0.05]. E Changes in the expression of Notch1 signalling pathway components following METH sensitization in the mPFC. There was significant downregulation of Notch1 [F _{(2, 17)} = 6.41, P < 0.01]_, Jagged1 [F _{(2, 15)} = 3.81, P < 0.05], RBP-J [F _{(2, 20)} = 18.05, P < 0.001], and Hes1 [F _{(2, 15)} = 29.68, P < 0.001] in both the METH-acute and METH_-sensitized groups by one-way ANOVA. F Representative images of immunofluorescence staining for Notch1 receptors in mPFC between the METH-sensitized group and the saline group. Quantitative analysis of Notch1-positive cells in the METH-sensitized group showed a significant decrease (t₂₆ = 9.76, ***P < 0.001) by student’s-t test (scale bar = 50 μm, n = 3 / group, 10–15 photos). G–K Intra-mPFC DAPT treatment attenuated METH-induced locomotor sensitization. G Procedure for the administration of DAPT in the mPFC in METH-induced sensitization mice. H Location of the DAPT and vehicle microinjection cannula tips in the mPFC. I Representative tracks of vehicle-METH and DAPT-METH mice on day 1 and day 23. J Intra-mPFC infusion of DAPT significantly attenuated the hyperlocomotion evoked by METH on day 1 and day 23. Mixed-design ANOVA with a LSD post hoc multiple comparison revealed significant main effects of DAPT [F _{(1, 21)} = 5.38, P < 0.05]; METH [F _{(1, 21)} = 206.21, P < 0.001]; but not DAPT×METH [F _{(1, 21)} = 1.36, P > 0.05]. K The time mice spent in the centre zone was higher in the DAPT-METH group than in the vehicle-METH group on day 23. Mixed-design ANOVA with LSD post hoc multiple comparisons show the main effect of DAPT [F _{(1, 20)} = 1.24, P > 0.05]; METH [F _{(1, 20)} = 0.74, P > 0.05] and DAPT × METH [F _{(1, 20)} = 6.45, P < 0.05]. *P < 0.05, **P < 0.01, ***P < 0.001 vs. paired saline group; ^P < 0.05 vs. paired METH group; #P < 0.05, ###P < 0.001 vs. same group on day 1. Data were presented as mean ± S.E.M, n = 6–8.

The specific methods of other behavioural testing, such as open filed test, novel object recognition (NOR), Y maze test, social interaction test (SIT), elevated plus maze (EPM), tail suspension test (TST) and forced swimming test (FST), were seen in the supplemental methods.