Fig 2: Compromising the protective effects of melatonin on Sev-associated toxicity by MT1 depletion. (A) Experimental design. (B, C) Western blotting and quantification of GluR1, GluR2, and PSD95 in WT and MT1 knockout mice treated with Sev or Sev-melatonin. (D) Immunostaining of vglut1. Melatonin did not exert protective effects on the expression of GluR1, GluR2, PSD95, and vglut1 in MT1-KO mice. (E, F) Western blotting and quantification of PDGFRa, Sox10, and Olig2 in WT and melatonin knockout mice treated with Sev or Sev-melatonin. (G) Immunostaining of Olig2. Melatonin did not exert significant effects on the expression of PDGFRa, Sox10, and Olig2 in MT1 knockout mice. N = 6 mice per group. Con, control. Sev, Sevoflurane. mel, melatonin. WT, wild type. KO, MT1 knockout. *p < .05. **p < .01. One-way ANOVA. Bars = 20 µm

Fig 3: Blockage of the protective effects of melatonin on Sev-induced synaptic impairment and dysmyelination by MT1-shRNA (shMT1). (A) Illustration of injection of lentivirus expressing shMT1. (B, C) Western blotting and quantification of MT1 in hippocampus injected with different shMT1. ShMT1-#2 was most effective in silencing the expression of MT1 (D) Western blotting and quantification of GluR1, GluR2, Homer, and PSD95 in mice of the following groups: control + vehicle, Sev + vehicle, Sev + mel, Sev + mel + EV (EV, empty vector), Sev + mel + shMT1. Notice that shMT1 blocked the up-regulation of GluR1, GluR2, Homer, and PSD95 by melatonin. (E) Immunostaining of vglut1. Notice the weak immunoreactivity of vglut1 in mice treated with shMT1 as compared with that in mice treated with EV. (F) Western blotting and quantification of PDGFRa, NG2, Sox10, and Olig2 in mice of the following groups: control + vehicle, Sev + vehicle, Sev + mel, Sev + mel + EV, Sev + mel + shMT1. Notice that shMT1 blocked the up-regulation of PDGFRa, NG2, Sox10, and Olig2 induced by melatonin. (G) Immunostaining of Olig2. N = 6 mice per group. Con, control. Sev, Sevoflurane. mel, melatonin. *p < .05. **p < .01. ***p < .001. One-way ANOVA

Fig 4: Relative loss of GABAergic synapses and Calbindin-immunoreactive cells. (a) Glutamatergic and GABAergic synapses were stained with Vglut1 (blue, white arrow) and Vgat (green, asterisk), respectively, for manual and automated quantification. Only synapses showing co-localization with Bsn (red) were counted. Confocal microscopy at 63×. (b) Processed immunofluorescence images for pre- and post-synapses of Fiji plug-in “Synapse Counter” and resulting co-localizations. Scale bars are 500 nm. (c) Relative quantification of GABAergic over glutamatergic synapses at 3 and 7 days after glutamate treatment as compared with time-matched sham-treated controls. (d,e) Relative amount of Calb1-positive neurons compared with Calb1-negative neurons in glutamate and sham-treated cultures after 7 days. Asterisks (*) indicate significance. ctrl—control, d—day, glu—glutamate.