Animals were assigned to one of the different treatment groups according to genotype, and compound or vehicle were administered orally by gavage. Animals were treated with MTC [3,7-bis(dimethylamino)phenothiazin-5-ylium chloride] or LMTX, either as the dihydrobromide [N,N,N’,N’-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen bromide)] or as the dihydromethane sulphonate [N,N,N’,N’-tetramethyl-10H-phenothiazine-3,7-diaminium bis(methanesulphonate)] salt form, as indicated (Fig. 1 ). Once administered within the body, the active moiety exists as a free base in redox equilibrium between MT+ and LMT (Schirmer et al., 2011 (link)). The dosages are expressed as weight of the compound inclusive of salt and extent of hydration and, for the different forms of MT, the amount of free MT base administered is given for each study. Thus, for certain experiments, the amount of free MT base may be up to 24% less for LMTX than for MTC.
Compounds were administered at a dose range of 5–75 mg/kg at an injection volume of 5 ml/kg body weight, administered daily in the morning (08.00–10.00 h). MTC (Simpsons, Gwent, UK) was dissolved in deionized water. Stock solutions were kept at −4°C in darkness for up to 7 days. LMTX (TauRx Therapeutics Ltd., Aberdeen, UK) was dissolved in argon-sparged deionized water and administered within 20 min of dissolution. The MT content was measured by HPLC after complete oxidation of the drug.
For behavioural observations of L1, we adopted a Monday–Friday gavaging regime over a period of up to 8 weeks (3 or 5 weeks before the test, 3 weeks during the test; Fig.2 a) for MTC and LMTX. For histopathological analysis, MTC and LMTX were administered consecutively for 19 days. By contrast, L66 mice were treated orally with daily injections for 19 successive days (14 days between the pretest and retest periods and 5 days during retest; Fig. 5 a). Cohort sizes for each drug condition under behavioural testing have been summarized in Table 1 . Between 24 and 48 h after the end of behavioural testing, the mice were killed by cervical dislocation; the brains were removed and snap frozen in liquid nitrogen for determination of MT content. Separate cohorts used for histological analysis of pathology without behavioural testing have been listed in Table 2 , and the mice in these cohorts were euthanized 1 h after the last injection. Animals were treated with an overdose of Euthatal; thereafter, their skulls were opened and their brains removed rapidly; the brains were stored in 4% paraformaldehyde, embedded in wax and processed as described (Melis et al., 2015 ).
Compounds were administered at a dose range of 5–75 mg/kg at an injection volume of 5 ml/kg body weight, administered daily in the morning (08.00–10.00 h). MTC (Simpsons, Gwent, UK) was dissolved in deionized water. Stock solutions were kept at −4°C in darkness for up to 7 days. LMTX (TauRx Therapeutics Ltd., Aberdeen, UK) was dissolved in argon-sparged deionized water and administered within 20 min of dissolution. The MT content was measured by HPLC after complete oxidation of the drug.
For behavioural observations of L1, we adopted a Monday–Friday gavaging regime over a period of up to 8 weeks (3 or 5 weeks before the test, 3 weeks during the test; Fig.