Additional Table 4.

Recent preclinical and clinical studies evaluating 3,4-methylenedioxymethamphetamine (MDMA)- and methylphenidate-induced brain dysfunctions and neurotoxicity

Drug	Species (n, sex)/age	Treatment/duration	Main findings	References
	C57BL/6J male mice (n=34), age N.A.	Saline or MDMA (10 mg/kg, two administrations per day, twice a week, i.p.) for 22,47, or 64 days.	The study found that MDMA elicited dopaminergic neurotoxicity in the CPu, SNc, and hippocampus, whose severity depended on the treatment regimen employed.	Costa et al., 2017
	Swiss-Webster male mice (sample size N.A.); 6-7 weeks old	Saline or MDMA (4 × 20 mg/kg, i.p.)	The study found that adolescent mice are more susceptible to neurotoxicity induced by acute MDMA compared to adult mice, due to greater metabolism of dopamine.	Chitre et al., 2020
	C57BL/6J male and female mice (n=45); 12 weeks old	Saline or MDMA (4 × 20 mg/kg, i.p.)	The study showed that MDMA causes sex-dependent dopaminergic neurotoxicity. Male mice had increased SOD 2 expression and reduced ubiquitin-proteasome system activity.	Costa et al., 2021
MDMA	Sprague-Dawley male rats (n=112); 5-6 weeks old	Pre-treatment: saline (s.c.) or caffeine (10 mg/kg, i.p.); treatment: saline (s.c.) or MDMA (5 mg/kg, s.c.) for 10 days	The study observed that rats exposed to MDMA during adolescence exhibited persistent and widespread dopaminergic neurotoxicity, leading to subsequent memory impairments at adulthood.	Cadoni et al., 2017
	Wistar male rats (n=33); 8-10 weeks old	Saline or MDMA (20 mg/kg, i.p.)	The study found that nitrosative stress from inducible nitric oxide synthase activity may play a pivotal role in mediating MDMA-induced neurotoxicity	Schiavone et al., 2019
	Male Cynomolgus monkeys (Macaca fascicularisi (n=14); 3-6 years old	Saline or MDMA (5 mg/kg, twice a day, s.c.) for 4 consecutive days.	The study unveiled that MDMA-induced neurotoxicity in monkeys extends beyond the serotonergic system to impact the dopaminergic system. Moreover, MDMA was found to exacerbate the dopaminergic neurotoxicity induced by MPTP.	Millot et al., 2020
	Formosan rock (Macaca cyclopis) monkeys (n=9, 3 females), 4-6 years old	Saline (s.c.), MDMA (5 mg/kg, twice a day, s.c.), or dextromethorphan (5 mg/kg, twice a day, s.c.) plus MDMA (5 mg/kg, twice a day, s.c.) for 4 days.	The study found that MDMA elicited long-lasting and region-specific serotonergic damage. In the hippocampus and amygdala, MDMA-induced neurotoxicity was partially contrasted by dextromethorphan.	Yeh et al., 2022
	Humans (n=16 healthy volunteers, 8 women); 20-27 years old	Placebo or MDMA (125 mg, p.o., corresponding to 1.8 ± 0.2 mg/kg body weight)	The study identified significant changes in the plasma levels of molecules associated with heightened stress, increased serotonergic activity, and enhanced inflammatory responses in individuals receiving MDMA.	Boxler et al., 2018
	Humans (n=39 healthy volunteers, 23 women; n=39 MDMA users, 22 women); 18-45 years old.	N.A.	The study revealed that the use of MDMA is not associated with white matter lesions. However, MDMA use was found to be associated with alterations in white matter diffusion, as evidenced by the heightened FA observed in multiple white tracts.	Zimmermann et al., 2022
	Wistar male rats (n=150); 8 weeks old	Saline, methylphenidate (10 mg/kg, i.p.), topiramate (10, 30, 50, 70 or 100 mg/kg, i.p.), methylphenidate plus topiramate for 14 or 21 days.	The study observed that methylphenidate triggered oxidative stress, inflammation, and neuronal damage, through the activation of NMDA, AMPA/kainate, GABA A, and Alpha 2 receptors. These effects were mitigated by topiramate administration.	Motaghinejad et al., 2017a,b
Methylphenidate	Sprague-Dawley male rats (sample size N.A.), PND 20 and 35	Saline, methylphenidate (2 mg/kg, twice a day, i.p.) for 14 days	The study discovered that chronic, but not acute, methylphenidate treatment alters in catecholaminergic targets in the parietal cortex and ventral CPu of young rats.	Quansah and Zetterström, 2019
	Wistar male rats (n=70); age N.A.	Saline, methylphenidate (10 mg/kg, i.p.), minocycline (10, 20, 30, or 40 mg/kg, i.p.), methylphenidate plus minocycline for 21 days.	The study found that minocycline treatment counteracted methylphenidate-induced toxic effects in the hippocampus, likely via the activation of the pCREB/BDNF or Akt/GSK3β pathways.	Motaghinejad and Motevalian, 2022

The articles reported in the table were arranged by model/species (cells, mice, rats, monkeys, humans) and in chronological order. AMPA: α-ammo-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF: brain-derived neurotrophic factor; CPu: caudate-putamen nucleus; FA: fractional anisotropy; GABA: γ-aminobutyric acid; GSK3β: glycogen synthase kinase-3 beta; i.p.: intraperitoneal injection; MDMA: 3,4-methylenedioxymethamphetamine; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NMDA: N-methyl-D-aspartate; p.o.: per os; s.c.: subcutaneous injection; SERT: serotonin transporter; SNc: substantia nigra pars compacta; SOD: superoxide dismutases.