用于哺乳动物行为研究的多通道光遗传系统
Optogenetics combines genetic technology with optical methods to achieve high-speed and accurate regulation of neurons in living animals, cells, and tissues. Fiber photometry is a sensitive and simple method to stimulate and record neuronal activity in the deep brains of animals. Multimode fibers can transmit stimulus light to neurons and collect their fluorescence. However, some animal behaviors result from the comprehensive action of the entire brain neural network, and the small size of the multimode fiber end face limits large-scale neuroscience research. Currently, techniques that can be used to evaluate large-scale animal neurodynamics are still limited. The previously proposed multi-channel optogenetic systems face three possible problems: a low degree of freedom of parameter adjustment, the need for independent customization of multi-channel fibers, and system integration problems caused by multi-sensors. In this paper, we report a multi-channel optogenetic system. The number, order, frequency, duty cycle, and other parameters of the multi-channel fibers can be independently adjusted by targeting specific fiber channels with a galvanometer. The system includes 1-to-7 fan-out multimode fiber bundles and only a scientific complementary metal-oxide-semiconductor (sCMOS) camera as the optical sensor, which effectively reduces the difficulty of the system integration. It provides a multi-channel, independent, flexible, and highly integrated solution for multi-channel optogenetic experiments.
In this study, 1-to-7 fan-out multimode fiber bundles are used. First, a scanning galvanometer is used to target a selected fiber channel, and the time-division multiplexing technology is used to modulate lasers with different wavelengths. Subsequently, a beam with a selected wavelength is coupled to a selected fiber channel for the experiments. The fluorescence signal collected by the fiber channel is then imaged by the sCMOS camera to achieve multi-channel fluorescence recording. System debugging, instrument control, and data processing are then integrated into an automatic software system to simplify the operation process of the system and meet the requirements of the multi-channel, multiwavelength, and multifunctional optogenetics experiments. In the next step, stability and crosstalk experiments are carried out on the multi-channel optogenetic system to evaluate the uniformity and independence of the multi-channel fibers. Additionally, typical biological experiments are performed to prove the feasibility of the system in optogenetic experiments.
The multi-channel optogenetic system has excellent parameter accuracy. The scanning galvanometer can accurately target the selected beam to a selected 200-µm diameter fiber channel. The test results of the fiber output frequency show that the average error of the stimulation frequency is 0.1% within the range of 5-500 Hz. Affected by the scanning angle of the galvanometer and the uniformity of illumination, the output powers of 7 channels are slightly different, but it still has excellent power stability, and the fluctuation does not exceed 6.02% (Fig. 3). The multi-channel crosstalk experiment results show that the strong stimulation light does not interfere with the fluorescence signals collected by other channels, and the fluorescence crosstalk between channels can be ignored (Fig. 4). Therefore, each channel of the multi-channel optogenetic system has excellent anti-interference capability, which helps control noise and ensure the accuracy of the experimental results. Finally, compared to the control mice, the photostimulation of the target region can induce unilateral rotation in mice, and the statistical results show more than twice the difference (Fig. 5). The experimental results show that the power value of every channel of the system is the same. Considering the microsecond response time of the scanning galvanometer, this system has the potential in special application scenarios such as multi-channel alternate outputs and ultrafast channel scanning.
In this study, a multi-channel optogenetic system is designed, which includes a laser time-division multiplexing module, galvanometer scanning module, and 1-to-7 fan-out multimode fiber bundles. The system solves problems in previous multi-channel systems: inflexible parameter adjustment, difficult customization of multi-channel fibers, and difficult system integration caused by a large number of instruments. The system can be used in the dynamics at multiple sites across the mammalian brain. The galvanometer scans different fiber channels at a frequency of 1 kHz. The output power of a single fiber can be flexibly adjusted within the range of 0-40 mW. Additionally, this system can flexibly adjust the wavelength, frequency, duty cycle, channel number, and channel sequence. Every channel has high frequency accuracy and power stability. The software system that integrates the control, algorithm, and analysis also significantly reduces the difficulty of system operation, which provides a convenient tool for research on the functional organization and behavior-related dynamics of mesoscale circuits in the brain. In the future, a correction function can be added to realize the real-time detection of physical parameters and closed loop-automatic experiments during operation, and the weight burden of the multi-channel fiber on the head of mice can be reduced by combining lightweight technology, providing a more miniaturized and intelligent solution for the multi-channel optogenetics field.
1 引言
光遗传学[1-5]将遗传技术与光学方法相结合,在细胞、组织、活体动物中实现对神经元的高速精准调控。近年来该技术被广泛应用于高分辨神经调控和多脑区同步实验,为病理机制探索和疾病治疗研究提供了支持[3,6-9]。光遗传学通常使用多模光纤(MMF)传输光[10-12],但多模光纤尺寸较小,限制了神经元编解码的区域范围。而动物的某些行为是整个大脑神经元网络综合作用的结果,目前,可用于评估动物大规模神经动力学的技术仍然有限[13-15]。动物记忆、焦虑、睡眠等复杂行为是大脑不同区域的神经元在执行特定任务期间作出的协调反应,需要多根光纤对不同类型的神经元或不同脑区进行同步实验[16-18]。此外,多只动物的特定行为研究也需要进行多光纤通道同步测试,例如研究社交互动的神经相关性[19-21]。因此,多通道、多波长、多功能的光遗传系统将成为神经环路与复杂行为研究的有效工具,为光遗传学实验探究提供了更多的选择。
近年来,研究者对多通道光遗传技术愈发关注。Adelsberger等[22]开发了双通道记录系统,通过增加光学传感器的数量,同时记录了两个脑区的神经活动,并证明小鼠运动皮层的不同子区控制着特定的运动。Paukert等[23]使用电荷耦合器件(CCD)相机成功记录了多通道光纤束的钙信号,在小鼠的不同脑区均发现了钙离子的激活信息。Kim等[24]开发了投影式独立光纤光度法,它可以同时记录来自多个脑区的荧光信号,成功量化了社交行为期间各个脑区的实时活动联系。Guo等[19]设计了可长期植入的高密度光纤阵列,该阵列能够实现哺乳动物大脑中跨区域的光纤光度记录。多通道光遗传系统通常利用大面积照明同时点亮所有光纤通道,并结合多个光学传感器独立采集各通道信号,但大部分多通道光纤及连接件需要定制,这给各个光纤通道参数的独立调整、通道数目的增减、系统的集成和多通道光纤的推广应用带来不便,在一定程度上限制了多通道光遗传实验的自由度。
本文采用扫描振镜来快速切换和精准靶向特定的光纤通道,通过优化扫描方案,可实现光刺激过程中多通道光纤数目、次序、频率、占空比等参数的独立调整;选取MMF定制1转7扇出多模光纤束,使用一台科学级互补金属氧化物半导体(sCMOS)相机作为光学传感器,有力降低了系统的集成与推广难度。可通过振镜扫描灵活选择实验通道,各个光纤通道参数稳定,串扰极小。小鼠光遗传实验表明,该系统稳定、灵活且易于使用。
2 系统开发
2.1 光学系统设计
图 1. 多通道光遗传系统示意图。(a)光学系统设计图;(b)1转7扇出多模光纤束及光纤入射端图像;(c)振镜电压对光斑位置的影响;(d)刺激光输出频率与程序设定频率的测试结果
Fig. 1. Schematics of multi-channel optogenetic system. (a) Design drawing of optical system; (b) 1-to-7 fan-out multimode fiber bundles and fiber incident end image; (c) influence of galvanometer voltage on spot position; (d) test results of stimulation light output frequency and program setting frequency
2.2 自动程序开发
图 2. 多通道光遗传系统控制方案。(a)系统的多样性实验方案;(b)灵活的光纤通道选择与次序设定;(c)软件界面;(d)双色荧光记录结果
Fig. 2. Control scheme of multi-channel optogenetic system. (a) Diversity experiment scheme of system; (b) flexible fiber channel selection and sequence setting; (c) software interface; (d) results recorded double-color fluorescence
3 结果与讨论
3.1 系统稳定性
在多通道实验探究中,保持各个通道功率的一致性至关重要,因此我们对每个通道的功率值进行了可靠性研究,以验证系统的稳定性与准确性。1转7扇出多模光纤束能传导刺激光和记录光,也能收集生物荧光信号。我们分别测试了光遗传刺激和信号记录时7个光纤通道的功率稳定性(标准差与均值的比值)。光刺激通常需要mW级别较高的功率[25],0~90 mW激光功率梯度下各个光纤通道的输出功率测试结果如
图 3. 多通道光纤功率稳定性及透过率。(a)多通道光纤端面图像及通道编号;(b)不同功率梯度下589 nm刺激光7通道功率稳定性;(c)不同功率梯度下473 nm记录光7通道功率稳定性;(d)不同厚度小鼠离体脑片的透过率
Fig. 3. Multi-channel fiber power stability and transmittance. (a) Multi-channel fiber end face image and channel No.; (b) 7-channel power stability at 589 nm stimulation light under different power gradients; (c) 7-channel power stability at 473 nm recording light under different power gradients; (d) transmittance of mouse in vitro brain slices with different thicknesses
除了光学参数的一致性外,光纤通道之间没有串扰也是关键[13,24]。串扰是由较强刺激光的反射和光纤通道间的荧光扩散造成的。选择1、2、3、4号光纤[
图 4. 不同光纤通道的串扰测试。(a)相机基底噪声;(b)不同模式下多通道串扰测试示意图;(c)单通道刺激时的双通道串扰测试结果;(d)图4(c)局部放大;(e)PL脑区单通道刺激时周围通道的串扰评估结果;(f)VTA脑区单通道刺激时周围通道的串扰评估结果
Fig. 4. Crosstalk test of different fiber channels. (a) Camera base noise; (b) schematics of multi-channel crosstalk test under different modes; (c) test result of two-channel crosstalk during single-channel stimulation; (d) local magnification of Fig.4(c); (e) crosstalk evaluation results of peripheral channels during single-channel stimulation in PL brain region; (f) crosstalk evaluation results of peripheral channels during single-channel stimulation in VTA brain region
3.2 行为学实验
我们利用多通道光遗传系统对Thy1-Cre转基因小鼠进行了光遗传干预。小鼠VTA脑区注射荧光蛋白ChrimsonR和钙指示剂GCaMP6s,注射3周后对小鼠进行单根光纤埋置,待小鼠术后恢复2周进行行为学实验,同时采用植入光纤但未表达光敏蛋白的小鼠作为对照。用功率为5 mW、频率为40 Hz的589 nm脉冲光(占空比为1∶9,持续时间为10 s)刺激VTA脑区,可以诱使小鼠发生单侧偏转运动。多次实验(N为重复实验次数)显示:小鼠单侧偏转运动均在脉冲光停止时结束,之后进行无规律运动[
图 5. 小鼠行为学实验图像。(a)小鼠VTA脑区的行为学实验;(b)有无光刺激下小鼠行为学实验中的运动轨迹;有无光刺激下小鼠(c)运动距离、(d)速度、(e)角度、(f)头部角度的统计结果
Fig. 5. Mice behavior experiment images. (a) Behavior experiment of mice VTA brain region; (b) movement tracks in mice behavior experiment with or without stimulation light; statistical results of (c) travelling distance, (d) speed, (e) angle, and (f) head angle of mice with or without stimulation light
4 结论
提出了一种多通道光遗传系统,能够支持7通道的光遗传研究,可灵活调整功率、频率、通道数目、通道次序和刺激模式,各个通道具有良好的稳定性。集控制、算法、分析于一体的软件系统也极大降低了系统的操作难度,为哺乳动物大脑中复杂环路的功能组织模式和行为相关动力学提供了有效的研究工具。未来可通过加入校正功能来实现物理参数的实时检测和闭环自动实验,结合轻量化技术减少多通道光纤重量对小鼠头部造成的负担,为多通道光遗传领域提供更加小型化、智能化的解决方案。
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Article Outline
杜吉超, 朱玥, 龚薇, 斯科. 用于哺乳动物行为研究的多通道光遗传系统[J]. 中国激光, 2023, 50(9): 0907302. Jichao Du, Yue Zhu, Wei Gong, Ke Si. Multi‑Channel Optogenetic System for Mammalian Behavior Research[J]. Chinese Journal of Lasers, 2023, 50(9): 0907302.