Optical Stimulation of Zebrafish Hair Cells Expressing Channelrhodopsin-2

Vertebrate hair cells are responsible for the high fidelity encoding of mechanical stimuli into trains of action potentials (spikes) in afferent neurons. Here, we generated a transgenic zebrafish line expressing Channelrhodopsin-2 (ChR2) under the control of the hair-cell specific myo6b promoter, in order to examine the role of the mechanoelectrical transduction (MET) channel in sensory encoding in afferent neurons. We performed in vivo recordings from afferent neurons of the zebrafish lateral line while activating hair cells with either mechanical stimuli from a waterjet or optical stimuli from flashes of ∼470-nm light. Comparison of the patterns of encoded spikes during 100-ms stimuli revealed no difference in mean first spike latency between the two modes of activation. However, there was a significant increase in the variability of first spike latency during optical stimulation as well as an increase in the mean number of spikes per stimulus. Next, we compared encoding of spikes during hair-cell stimulation at 10, 20, and 40-Hz. Consistent with the increased variability of first spike latency, we saw a significant decrease in the vector strength of phase-locked spiking during optical stimulation. These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli. Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae. In transgenic larvae, 100-ms flashes of ∼470-nm light resulted in escape responses that occurred concomitantly with field recordings indicating Mauthner cell activity. Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.

脊椎动物毛细胞负责将机械刺激高保真地编码为传入神经元内的动作电位（锋电位）序列。本研究构建了一株在毛细胞特异性myo6b启动子调控下表达光敏感通道蛋白-2（Channelrhodopsin-2, ChR2）的转基因斑马鱼品系，旨在探究机械电转导（mechanoelectrical transduction, MET）通道在传入神经元感觉编码中的作用。我们对斑马鱼侧线传入神经元开展在体记录，分别采用水流产生的机械刺激和约470纳米波长闪光的光刺激激活毛细胞。对比100毫秒刺激期间编码的锋电位模式发现，两种激活方式下首次锋电位潜伏期的平均值无显著差异。但光刺激条件下，首次锋电位潜伏期的变异度显著升高，且每次刺激的平均锋电位数量增加。随后，我们比较了10、20和40赫兹频率下毛细胞刺激的锋电位编码情况。与首次锋电位潜伏期变异度升高的结果一致，光刺激时锋电位锁相的矢量强度显著降低。上述在体实验结果证实，MET通道在机械感觉刺激编码过程中，对维持首次锋电位潜伏期的高保真度具有生理功能。最后，我们探究了通过ChR2激活实现毛细胞远程激活，是否足以诱导自由游动幼体产生逃逸反应。在转基因幼体中，约470纳米的100毫秒闪光可引发逃逸反应，同时场电位记录显示伴随莫特纳细胞（Mauthner cell）活动。综上，myo6b:ChR2转基因品系为研究毛细胞功能与感觉编码、毛细胞向莫特纳细胞的感觉输入，以及在自由游动斑马鱼中远程诱发行为的能力提供了研究平台。