BACKGROUND A fundamental limit to the study of the peripheral nervous system and its effect on organ function is the lack of tools to selectively target and stimulate specific neurons. Traditional implant and electrode-based systems remain too large and invasive for use at the organ or sub-organ level (without stimulating or effecting neighboring organs and tissues). Recent progress in optical and genetic tools (such as optogenetics) has provided a new level of molecular specificity and selectivity to the neurons that are stimulated by bioelectronic devices. However, the modified neurons that result from use of these tools (that can be selectively activated based on expression of light, heat, or stimuli sensitive ion channels) often still require stimulation by implantable devices and face difficult scientific, technical, and regulatory hurdles for clinical translation. NEW METHOD Herein, we present a new tool for selective activation of neuronal pathways using anatomical site-specific, peripheral focused ultrasound neuromodulation (pFUS). RESULTS We utilize three experimental models to expand upon and further characterize pFUS beyond data outlined to our initial report (Cotero et al., 2019a), and further demonstrate its importance as a new investigative and translational tool. First, we utilized an interconnected microporous gel scaffold to culture isolated dorsal root ganglion (DRG) neurons in an interconnected, three-dimensional in vitro culture. (Griffin et al., 2015, Tay et al., 2018) Using this system, we directly applied ultrasound (US) stimuli and confirmed US activation of peripheral neurons at pressures consistent with recent in vivo observations. (Cotero et al., 2019a, Zachs, 2019, Gigliotti et al., 2013) Next, we tested the capability of pFUS to activate previously reported nerve pathways at multiple locations within the neural circuit, including primary sensory ganglia (i.e. inferior ganglion of the vagus nerve), peripheral ganglia (i.e. sacral ganglia), and within target end-organs. In addition, we compared selective activation of multiple anatomically overlapping neural pathways (i.e. activation of the cholinergic anti-inflammatory pathway (Tracey, 2009, Pavlov and Tracey, 2012) vs. metabolic sensory pathways (O'Hare and Zsombok, 2015, Roh et al., 2016, Pocai et al., 2005) after stimulation of each separate target site. Finally, we utilized an established model of metabolic dysfunction (the LPS-induced inflammation/hyperglycemia model) to demonstrate pFUS capability to stimulate and assess alternative therapeutic stimulation sites (i.e. liver, pancreas, and intestines) in a simple and clinically relevant manner. This is demonstrated by ultrasound induced attenuation of LPS-induced hyperglycemia by stimulation at all three anatomical targets, and mapping of the effect to a specific molecular product of excitable cell types within each stimulus site. COMPARISON WITH EXISTING METHODS The ease-of-use and non-invasive nature of pFUS provides a solution to many of the challenges facing traditional toolsets, such as implantable electrodes and genetic/optogenetic nerve stimulation strategies. CONCLUSIONS The pFUS tool described herein provides a fundamental technology for the future study and manipulation of the peripheral nervous and neuroendocrine systems.

中文翻译：

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周边聚焦超声神经调节（pFUS）。

背景技术对周围神经系统及其对器官功能的影响的研究的基本局限性是缺乏选择性地靶向和刺激特定神经元的工具。传统的基于植入物和电极的系统仍然太大且具有侵入性，无法在器官或亚器官水平使用（不会刺激或影响邻近的器官和组织）。光学和遗传工具（例如光遗传学）的最新进展为生物电子设备刺激的神经元提供了更高水平的分子特异性和选择性。但是，由于使用了这些工具而产生的修饰神经元（可以根据光，热或刺激敏感离子通道的表达而选择性激活），仍然需要植入式设备进行刺激，并且面临着科学，技术，和临床翻译的监管障碍。新方法本文中，我们介绍了一种使用解剖部位特异性，外周聚焦超声神经调节（pFUS）选择性激活神经元通路的新工具。结果我们利用三种实验模型来扩展pFUS并进一步对其进行表征，超出了初次报告中概述的数据（Cotero等，2019a），并进一步证明了其作为新的研究和翻译工具的重要性。首先，我们利用互连的微孔凝胶支架在互连的三维体外培养物中培养分离的背根神经节（DRG）神经元。（Griffin等人，2015，Tay等人，2018）使用该系统，我们直接施加了超声（US）刺激并确认了US激活周围神经元的压力与近期体内观察结果一致。（Cotero et al。，2019a，Zachs，2019，Gigliotti et al。，2013）接下来，我们测试了pFUS激活先前报道的在神经回路中多个位置的神经通路的能力，包括初级感觉神经节（即下神经节）。迷走神经），周围神经节（即神经节）和目标终末器官内。此外，我们比较了多种解剖学重叠神经通路的选择性激活（即胆碱能抗炎通路的激活（Tracey，2009； Pavlov和Tracey，2012））与代谢感觉通路（O'Hare和Zsombok，2015，Roh等）的比较。等人，2016年，Pocai等人，2005年）刺激了每个单独的目标位置。我们利用已建立的代谢功能障碍模型（LPS诱导的炎症/高血糖模型）来证明pFUS以简单且与临床相关的方式刺激和评估其他治疗性刺激部位（即肝脏，胰腺和肠）的能力。这通过超声刺激所有三个解剖学目标刺激脂多糖诱导的高血糖减轻，以及在每个刺激部位内将作用映射到可兴奋细胞类型的特定分子产物上来证明。与现有方法的比较pFUS的易用性和非侵入性为解决传统工具集所面临的许多挑战提供了解决方案，例如植入式电极和遗传/遗传神经刺激策略。