To investigate the involvement of oxidative stress in Ppp1r2-cre/fGluN1 KO mice, cortical ROS levels in brain sections from KO animals (8- and 16-week-old) were examined after i.p. injection of dihydroethidium (DHE). DHE has been used to monitor cortical ROS production in vivo (13), because DHE is oxidized by ROS, forming ethidium bromide, which emits red fluorescence once it intercalates with DNA (21). A prominent increase of cortical ROS level was observed in group-housed KO animals compared with fGluN1 or Ppp1r2-cre (Figure S1A, B) controls at 16 but not at 8 weeks old (Figure 1). However, PWSI sharply augmented ROS levels in mPFC and S1 cortex of KO mice both at 8 and 16 weeks of age. Overall, the oxidized DHE signals were neither excitatory nor inhibitory neuron-specific, as assessed by excitatory neuronal marker neurogranin and various interneuron markers, such as parvalbumin, calretinin, and calbindin (Figure S1C in Supplement 1).
