Application of endogenous autofluorescence signal for functional brain imaging

我们开发了一种神经成像的方法，利用活动依赖变化的绿色荧光的黄蛋白被蓝光激发。我们观察到内源性绿色荧光增加响应电刺激在大鼠听觉皮层切片。在室温下，重复电刺激开始后2 ~ 3 s观察到的峰值增幅高达30%左右。在无钙、无葡萄糖、无氧培养基灌注的切片中，也未见这种现象。这些数据表明，绿色荧光反应归因于参与线粒体能量代谢的黄蛋白的荧光。在用聚氨酯麻醉的大鼠中，我们观察到声音刺激后听觉皮层暴露表面的绿色荧光变化的清晰图像。这些荧光变化是在没有使用平均法的情况下观察到的，因为它们比由心跳或呼吸引起的光学噪声大得多。我们还试图利用黄蛋白荧光的波动来研究自发神经活动之间的空间关联模式。在覆盖琼脂的正常体感觉皮层中，呼吸运动校正后的荧光信号在ROI周围呈正相关。在ROI周围施加强电刺激增加了ROI周围正相关的程度。该方法还用于观察学习引起的大鼠初级体感皮层神经活动的变化。一只缺水的大鼠被训练来辨别斯金纳箱中地板振动的频率（20赫兹或40赫兹）。在连续3-5天完成这种区分学习后，所有被试大鼠对奖励频率的体感神经反应都大于对未奖励频率的反应。在小鼠中，小鼠的透明颅骨允许对皮质活动进行经颅自体荧光成像。少

We developed a method of neural imaging using activity-dependent changes in green fluorescence of flavoproteins excited by blue light. We observed an increase in endogenous green fluorescence in response to electrical stimulation in the slices of the rat auditory cortex. The amplitude of the peak increase, which was observed 2-3 s after the initiation of the repetitive electrical stimulation, was as large as about 30% at room temperature. They were also not observed in the slices perfused with medium lacking calcium, glucose or oxygen. These data indicate that the green fluorescent responses are attributed to the fluorescence of flavoproteins involved in the energy metabolism in mitochondria. In anesthetized rats with urethane, we observed clear images of the green fluorescent changes on the exposed surface of the auditory cortex following sound stimuli. These fluorescent changes were observed without using averaging, since they were much larger than the optical noises caused by heartb … More eats or breathing. We also tried to investigate spatial pattern of correlation between spontaneous neural activites using fluctuations in flavoprotein fluorescence. In the normal somatosensory cortex covered with agar, positive correlation around a ROI was observed in the fluorescence signals after correction for breathing movements. Tetanic stimulation applied around the ROI increased the magnitude of the positive correlation around the ROI. This method was also used to visualize learning-induced changes of neural activities in the rat primary somatosensory cortex. A water-deprived rat was trained to discriminate the frequency (20 Hz or 40 z) of floor vibration in a Skinner box. After this discrimination learning was achieved in consecutive 3-5 days, the somatosensory neural responses to the rewarded frequency were larger than that in response to the unrewarded frequency in all of the rats tested. In mice, the transparent skull of mice allowed trans-cranial autofluorescence imaging of cortical activites. Less