Fig. 1. (A, B) 12-hr Amount and episode duration of NREM sleep during the light phase after ablation of VM neurons. (C, D) Decreased power of sleep spindles on the parietal EEG but not in the frontal EEG during the light phase after ablation of VM neurons. **p<0.01, paired t-test. Data are presented as mean ± SEM.Fig. 2. (A) Examples of decreased signals of Calb1 immunostaining in the VM after ablation of VMCalb1 neurons. (B) Quantitative results of Calb1 immunostaining. ****p<0.0001, t-test. Data are presented as mean ± SEM.Histological verification of the VMCalb1-ablated miceNext, I aimed to examine whether the Calbindin 1-positive neurons in the VM (VMCalb1 neurons), which project their axons to all the cortical areas, are responsible for regulating normal sleep spindles. Cre-dependent Caspase-3 proteins are virally expressed in the VMCalb1 neurons in Calb1-Cre mice to trigger programmed cell death, which results in ablation of VMCalb1 neurons in the mice (VMCalb1-ablated mice). To confirm the VMCalb1 ablation, Calb1 immunostaining was performed with the brain slices containing the VM (Fig. 2A), and the signals of VMCalb1 neurons were quantified. As a result, the percentage of the area containing Calb1 signals in the VM was diminished, indicating VMCalb1 neurons were ablated in the VMCalb1-ablated mice (Fig. 2B).Decreased sleep spindles in the frontal cortex of the VMCalb1-ablated miceNext, EEG was recorded, and sleep-related phenotypes were examined for 2 weeks after the AAV injection.― 258 ―
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