利用DeepLabCut-Live在闭环光遗传刺激下探测行为状态依赖的神经回路

IF 2.7 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-05-26 DOI:10.1016/j.jneumeth.2025.110495

Melissa Gonzalez , Mark A. Gradwell , Joshua K. Thackray , Kanaksha K. Temkar , Komal R. Patel , Victoria E. Abraira

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引用次数: 0

摘要

闭环行为范式允许实时调查状态相关的神经回路背后的行为。然而，由于实时操纵脊髓回路的分子工具和技术的限制，研究情境依赖的运动扰动具有挑战性。我们开发了一种新的闭环光遗传刺激模式，利用DeepLabCut-Live姿势估计来操纵小鼠运动周期特定阶段的主要感觉传入活动。训练了一个紧凑的DeepLabCut模型来实时跟踪后肢运动，并将其集成到Bonsai可视化编程框架中。该系统使LED触发的光刺激的感觉神经元表达通道视紫红质基于用户定义的姿势为基础的标准，如立场或摇摆阶段。结果在跑步机运动过程中，伤害性TRPV1+感觉神经元的光遗传激活可靠地诱发了爪退缩反应。在站立阶段的刺激产生了短暂的退出，并影响了接下来的摇摆阶段的持续时间。在摇摆阶段的刺激增加了摇摆期间的爪收回高度，并减少了接下来的站立阶段的持续时间。与现有方法的比较该方法在基于运动相位的脊髓回路操作中具有较高的时空精度。与以前的方法不同，这种闭环系统考虑了感觉运动反应的状态依赖性质，实现了对运动的控制和实时调制。结论将DeepLabCut-Live与光遗传学相结合，为解剖感觉反馈和脊髓中间神经元在运动中的情境依赖作用提供了有力的工具。该技术为揭示状态依赖行为的神经基础开辟了新的途径，并在基于姿态估计的实时闭环操作研究中具有广泛的适用性。

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Using DeepLabCut-Live to probe state dependent neural circuits of behavior with closed-loop optogenetic stimulation

Background

Closed-loop behavior paradigms allow for real-time investigation of state-dependent neural circuits underlying behavior. However, studying context-dependent locomotor perturbations is challenging due to limitations in molecular tools and techniques for real-time manipulation of spinal circuits.

New method

We developed a novel closed-loop optogenetic stimulation paradigm that leverages DeepLabCut-Live pose estimation to manipulate primary sensory afferent activity at specific phases of the locomotor cycle in mice. A compact DeepLabCut model was trained to track hindlimb kinematics in real-time and integrated into the Bonsai visual programming framework. This system enabled LED triggered photo-stimulation of sensory neurons expressing channelrhodopsin based on user-defined pose-based criteria, such as stance or swing phase.

Results

Optogenetic activation of nociceptive TRPV1⁺ sensory neurons during treadmill locomotion reliably evoked paw withdrawal responses. Stimulation during the stance phase generated a brief withdrawal and impacted the duration of the following swing phase. Stimulation during the swing phase increased the height of paw withdrawal during swing and reduced the duration of the following stance phase.

Comparison with existing methods

This method allows for high spatiotemporal precision in manipulating spinal circuits based on locomotor phase. Unlike previous approaches, this closed-loop system accounts for state-dependent nature of sensorimotor responses, enabling controlled, real-time modulation of locomotion.

Conclusions

Integrating DeepLabCut-Live with optogenetics provides a powerful tool for dissecting the context-dependent role of sensory feedback and spinal interneurons in locomotion. This technique opens new avenues for uncovering the neural substrates of state-dependent behaviors and has broad applicability for studies of real-time closed-loop manipulation based on pose estimation.

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.