Adult male Sprague-Dawley (SD) rats (280–300 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed in groups of 4 per cage after arrival with appropriate temperature (22 ± 2°C) and humidity (50 ± 5%), as well as freely accessible water and food. The lighting time was controlled, under a 12-h light/dark cycle. All behavioral experiments were performed during the animal’s dark period. Animal care and experimentation were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Biomedical Ethics Committee for Animal Use and Protection of Peking University (No: LA2019067).

The METH and 3-MMC were provided by Drug Intelligence and Forensic Center of Ministry of Public Security, China. Both drugs were dissolved in 0.9% saline and ready for intraperitoneal injection.

The conditioned place preference (CPP) procedure was performed using an unbiased, counterbalanced protocol that has been described previously (Liang et al., 2017). The apparatus for CPP conditioning consisted of 10 identical three-chamber polyvinyl chloride (PVC) boxes. The boxes had two larger chambers (27.9 cm length × 21.0 cm width × 20.9 cm height) that differed in their floor texture (bar or grid, respectively) and the houselights on the walls. The two larger chambers were separated by a smaller chamber (12.1 cm length × 21.0 cm width × 20.9 cm height, with a smooth PVC floor). Baseline preference was assessed by placing the rats in the center chamber of the CPP apparatus and allowing them to explore all three chambers freely for 15 min. Rats that showed a strong unconditioned preference for either side chamber (i.e., >500 s) were excluded from the experiments. On the following 1, 3, 5, and 7 days, the rats received saline, 3-MMC (1, 3, or 10 mg/kg, i.p.), or METH (1 mg/kg, i.p.). Based on the dose of 3-MMC at 0.3 mg/kg for pigs (as 2 mg/kg for rats) in a pharmacological study (Shimshoni et al., 2015; Nair and Jacob, 2016) and intraperitoneal injection of 4-methylethcathinone at 1, 3, or 10 mg/kg in another study (Xu et al., 2016), we chose the dose above for CPP training. All rats received saline injection on the 2, 4, 6, and 8 days. After each injection, rats were immediately confined to the drug-paired or saline-paired conditioning chamber for 45 min before being returned to their home cages. On days 9, rats were placed in the central compartment without receiving any injection and were allowed to explore the entire apparatus freely for 15 min. The CPP score was calculated by the time (in seconds) spent in the drug-paired chamber minus the time spent in the saline-paired chamber during the CPP tests.

Locomotor Activity Test was conducted based on previous study (Deng et al., 2017). All rats were habituated to the locomotor chambers (40 cm × 40 cm × 65 cm) for 3 days (120 min/day) before locomotor activity test. In test day, we first recorded locomotor activity for 30 min, then rats were injected with a single of saline, 3-MMC (1, 3, or 10 mg/kg) or METH (1 mg/kg) and continued to record for 120 min. All locomotor activities were recorded and analyzed with an automated video tracking system (DigBehv-LM4; Shanghai Jiliang Software Technology, Shanghai, China). Locomotor activity is expressed as the total distance traveled in centimeters during a predetermined period of time.

The elevated plus maze (EPM) was utilized to evaluate anxiety-like behavior based on the rats’ natural fear of open, unprotected, and elevated spaces (Pellow et al., 1985). The EPM consisted of four crossed narrow arms elevated 70 cm from the floor, with two open arms and two closed arms (50 cm long and 10 cm wide). 10 min after acute injection (saline, 3-MMC [1, 3 or 10 mg/kg], or METH [1 mg/kg]) or the last injection of chronic drug administration, each rat was placed in the central zone of the EPM with its head facing an open arm and was allowed to freely explore the maze for 5 min. The time spent in each arm and the traces of rats were analyzed with the EthoVision XT (Noldus IT, Netherlands).

Brains were fixed with 4% paraformaldehyde (PFA) for at least 24 h and transferred into phosphate-buffered saline (PBS, pH 7.2) containing 10, 20, and 30% sucrose until they sank. Coronal sections of the brain were cut into 20 μm slice at −20°C in the cryostat (Leica, CM3050 S). Slices were washed with three times of PBS for 5 min, blocked with 5% bovine serum albumin (BSA) dissolved in 0.2% Triton X-100 for 1 h at room temperature and incubated with Rabbit anti-c-Fos (1:500, Abcam, ab190289) primary antibodies dissolved in blocking buffer overnight at 4°C, followed by four times of 15-min wash with PBS at room temperature. After that, slices were incubated with Goat anti-Rabbit Secondary Antibody (Alexa Fluor 488, 1:500, Invitrogen, A-11008) dissolved in blocking buffer for 2 h at room temperature followed by four times of 15-min wash with PBS. Lastly, the slices were mounted by DAPI (Abcam, ab285390) and stored at 4°C for analysis.

Fluorescent images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan) with a 20× objective lens and analyzed according to our previous study (Liang et al., 2017), in which at least three sections were selected from each brain region for each rat. The size of sampled areas for cell quantifications of each brain region from each section was 0.39 mm × 0.39 mm. Brightness and contrast adjustments were applied to the whole image. The number of c-Fos-positive cells were identified and counted in IMARIS software (Oxford Instruments, United Kingdom). An investigator blinded to the experimental conditions performed the image analyses.

The experiments were performed as previously described (Yu et al., 2022). Whole-cell patch-clamp recordings of NAc neurons were performed 24 h after the last injection of chronic administration with saline, 1 mg/kg METH or 3 mg/kg 3-MMC. The brains were rapidly removed after anesthetization and 250 μm coronal slices were prepared with a vibratome (Leica VT1200S) in ice-cold solution containing (in mM): 80 NaCl, 26 NaHCO₃, 3.0 KCl, 1.0 NaH₂PO₄, 1.3 MgCl₂, 1.0 CaCl₂, 20 D-glucose, and 75 sucrose, saturated with 95% O₂ and 5% CO₂. The slices were moved to an incubation chamber containing artificial cerebrospinal fluid (ACSF) consisted of the following (in mM): 124 NaCl, 26 NaHCO₃, 3.0 KCl, 1.0 NaH₂PO₄, 1.3 MgCl₂, 1.5 CaCl₂, 20 D-glucose, saturated with 95% O₂ and 5% CO₂ at 34°C for 30 min and then at room temperature until used for recording.

For sIPSC recording, the pipette solution contained the following (in mm): 120 CsCl, 20 TEA-Cl, 4 ATP-Mg, 0.3 GTP, 0.5 EGTA, 10 HEPES, and 4.0 QX-314 (pH 7.2, 270–280 mOsm with sucrose). For sEPSC recording, the pipette solution contained the following (in mm): 110 Cs methylsulfate, 15 CsCl, 20 TEA-Cl, 4 ATP-Mg, 0.3 GTP, 0.5 EGTA, 10 HEPES, and 4.0 QX-314 (pH 7.2, 270–280 mOsm with sucrose).

All signals were amplified (Multiclamp 700B, Axon Instruments), filtered at 5 kHz and digitized at 20 kHz (National Instruments Board PCI-MIO-16E4, Igor, Wave Metrics). Data were recorded within Axon pClamp 10 (Molecular Devices, CA, United States). Data analysis referred to our previous research (Yu et al., 2022).

Statistical analyses were performed with GraphPad Prism 9. One-way analysis of variance (ANOVA), two-way ANOVA, or paired t-test was used to analyze data when applicable. Bonferroni test was used for post hoc analysis after ANOVA. Data were presented as mean ± standard errors of the mean (SEM). Significance was defined as *P < 0.05, ^**P < 0.01, and ^***P < 0.001.

Conditioned place preference and locomotor activity are paradigms commonly employed to determine the rewarding and psychomotor properties of psychoactive drugs, respectively (Tzschentke, 2007). To study the rewarding effect of 3-MMC, the rats underwent CPP training (1, 3, 10 mg/kg of 3-MMC; or 1 mg/kg of METH) for 8 days and received CPP test on day 9. Rats with 3 mg/kg 3-MMC, 10 mg/kg 3-MMC and 1 mg/kg METH displayed increased CPP score (paired t-test: t₉ = 3.46, ^**P = 0.0072 for 1 mg/kg METH; t₉ = 2.99, *P = 0.0152 for 3 mg/kg 3-MMC; t₉ = 2.76, *P = 0.0222 for 10 mg/kg 3-MMC), implying that 3-MMC induced CPP dose-dependently (Figure 1A).

Because 3 mg/kg 3-MMC could effectively induce CPP, we next assessed its effect on locomotor activity of rats after acute exposure. A two-way ANOVA of moving distance revealed a main effect of drugs [F_(2,22) = 97.94, P < 0.0001], implying the psychomotor properties of 3-MMC. As shown in Figure 1B, rats treated with 3-MMC exhibited an enhanced locomotor activity from 5 to 30 min and 95–100 min (post hoc test: *P < 0.05 for 3-MMC versus saline), while rats with METH injection showed increased activity from 5 to 120 min (post hoc test: ^#P < 0.05 for METH versus saline).

To investigate the effect of acute 3-MMC exposure on anxiety-like behavior, we tested the performance of rats in EMP after acute 3-MMC (1, 3, and 10 mg/kg) injection. EMP test showed that rats with 3 mg/kg 3-MMC spent more time in the open arms (paired t-test: t₆ = 2.45, *P = 0.0497), implying the rapid anxiolytic-like effect of 3-MMC (Figure 2A). However, the anxiolytic-like effect of 3-MMC instead decreased when the concentration was increased to 10 mg/kg (paired t-test: t₆ = 2.00, P = 0.092). In addition, there was no change of performance in EMP before and after acute METH exposure (paired t-test: t₆ = 1.20, P = 0.2763).

We next tested the effect of repeated 3-MMC (3 mg/kg for seven consecutive days) and METH (1 mg/kg for seven consecutive days) exposure on anxiety-like behavior in rats. Result showed that rats administered with chronic 3-MMC spent less time in the open arms (paired t-test: t₈ = 3.223, *P = 0.0122), implying that prolonged use of 3-MMC could increase anxiety-like behavior (Figure 2C). And there was no change of performance in EMP before and after chronic METH exposure (paired t-test: t₈ = 0.95, P = 0.3698). All travel traces of rats in EMP test were presented in Figures 2B,D.

Because results above showed that the behavioral changes caused by 3 mg/kg 3-MMC were more significant, we investigated the alternations of neuronal activity after chronic exposure to 3-MMC under this dose. Rats received injection of drugs for 7 days and were sacrificed 90 min after the last injection without any behavioral test. Next, we examined c-Fos expression in brain regions which had been reported to be involved in drug addiction (Piazza and Deroche-Gamonet, 2013; Apaydin et al., 2018) to identify the specific regions in response to chronic 3-MMC exposure.

As shown in Figures 3A,B, rats with repeated 3-MMC injection presented increased number of c-Fos labeled neurons in anterior cingulate cortex (ACC), NAc, and ventral tegmental area (VTA) compared with saline group [two-way ANOVA revealed a significant interaction effect: F_(18,81) = 8.35, P < 0.0001, post hoc test: *P < 0.05 or ^**P < 0.01 for 3-MMC versus saline group in brain regions above]. Noteworthy, 3-MMC exposure induced more significant activation of VTA than METH (post hoc test: ^#P < 0.05 for 3-MMC versus METH), which presented increased expression of c-Fos in ACC, anterior insular cortex (aIC), NAc, central amygdala (CeA) and VTA (post hoc test: *P < 0.05 or ^**P < 0.01 for METH versus saline group in brain regions above). The data of statistics results were shown in Table 1.

As psychoactive substance, both 3-MMC and METH induced activation of NAc neurons after chronic exposure. However, there is no research about the effect of 3-MMC on NAc yet, a key structure in rewarding, motivation, and incentivized learning (Volkow et al., 2011). To determine the effects of 3-MMC on synaptic activity, we recorded spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in NAc. As shown in Figures 4E,F, the amplitude of sIPSCs was decreased after repeated 3-MMC exposure [one-way ANOVA, F_(2,81) = 7.97, P = 0.0007, post hoc test: *P < 0.05 for saline versus 3-MMC, ^***P < 0.001 for saline versus METH, P = 0.7895 for 3-MMC vs METH], while frequency of sIPSCs remained unchanged [one-way ANOVA, F_(2,81) = 3.02; P = 0.0541]. Neither the frequency [one-way ANOVA, F_(2,81) = 2.17; P = 0.1207] nor the amplitude of sEPSCs [one-way ANOVA, F_(2,81) = 2.97; P = 0.0569] was affected by repeated 3-MMC exposure (Figures 4B,C). The representative sEPSCs and sIPSCs traces of NAc neurons were presented in Figures 4A,D.