一种用于测量头部约束小鼠空间学习的开源虚拟现实系统
Summary
在这里，我们提出了一个简化的开源硬件和软件设置，用于使用虚拟现实（VR）研究鼠标空间学习。该系统利用微控制器网络和运行易于使用的Python图形软件包的单板计算机，向在滚轮上运行的头部受限鼠标显示虚拟线性轨迹。
Abstract
小鼠头部约束行为实验使神经科学家能够使用高分辨率电生理和光学成像工具观察神经回路活动，同时向行为动物提供精确的感官刺激。最近，使用虚拟现实（VR）环境的人类和啮齿动物研究表明，由于对空间和上下文线索等参数的极其精确的控制，VR是揭示海马体和皮层空间学习背后的神经机制的重要工具。然而，为啮齿动物的空间行为设置虚拟环境可能成本高昂，并且需要广泛的工程和计算机编程背景。在这里，我们提出了一个基于廉价，模块化，开源硬件和软件的简单而强大的系统，使研究人员能够使用VR环境研究头部约束小鼠的空间学习。该系统使用耦合的微控制器来测量运动并提供行为刺激，而头部约束的小鼠在轮子上奔跑，与单板计算机上运行的图形软件包呈现的虚拟线性轨道环境保持一致。对分布式处理的强调使研究人员能够设计灵活的模块化系统来引发和测量小鼠的复杂空间行为，以确定哺乳动物大脑中神经回路活动与空间学习之间的联系。
Introduction
空间导航是一种重要的行为，动物通过这种行为将新位置的特征编码到认知地图中，用于查找可能奖励的区域并避免潜在危险的区域。空间导航背后的认知过程与记忆有着千丝万缕的联系，它们在海马体1和皮层中共享一个神经基质，在这些区域中，这些区域的神经回路整合传入的信息，形成环境和事件的认知图谱，以供以后回忆2。虽然海马体3，4 中的位置细胞和内嗅皮层5 中网格细胞的发现揭示了海马体内认知图谱是如何形成的，但关于海马体的特定神经亚型、微电路和单个亚区（齿状回和角氨区，CA3-1）如何相互作用并参与空间记忆的形成和回忆，仍然存在许多问题。
体内双光子成像一直是揭示感觉神经生理学中细胞和群体动态的有用工具6，7;然而，头枕的典型必要性限制了这种方法在检查哺乳动物空间行为方面的实用性。虚拟现实（VR）8的出现解决了这一缺点，它提供了身临其境和逼真的视觉空间环境，而头部约束的小鼠在球或跑步机上奔跑，以研究海马体8，9，10和皮层11中的空间和上下文编码。此外，将VR环境与行为小鼠一起使用，使神经科学研究人员能够通过精确控制VR环境12的元素（例如，视觉流，上下文调制）来剖析空间行为的组成部分，这在现实世界的空间学习实验中是不可能的，例如莫里斯水迷宫，巴恩斯迷宫或孔板任务。
Visual VR 环境通常在计算机的图形处理单元 （GPU） 上渲染，该单元处理快速计算在屏幕上实时建模移动 3D 环境所需的数千个多边形的负载。较大的处理要求通常需要使用带有GPU的单独PC，该GPU将视觉环境渲染到显示器，多个屏幕13或投影仪14 ，因为从跑步机，轮子或动物下方的泡沫球记录运动。因此，由此产生的用于控制、渲染和投影 VR 环境的设备相对昂贵、笨重且笨重。此外，文献中的许多此类环境都是使用专有软件实现的，这些软件既昂贵又只能在专用PC上运行。
出于这些原因，我们设计了一个开源VR系统，使用Raspberry Pi单板计算机研究头部约束小鼠的空间学习行为。这款Linux计算机既小又便宜，但包含一个用于3D渲染的GPU芯片，允许将VR环境与各种个人设置中的显示器或行为设备集成。此外，我们还开发了一个用Python编写的图形软件包“HallPassVR”，它利用单板计算机通过重新组合使用图形用户界面（GUI）选择的自定义视觉特征来渲染简单的视觉空间环境，虚拟线性轨道或走廊。它与微控制器子系统（例如 ESP32 或 Arduino）相结合，以测量运动和协调行为，例如通过提供其他形式的感官刺激或奖励来促进强化学习。该系统提供了一种廉价、灵活且易于使用的替代方法，用于在双光子成像（或其他需要头部固定的技术）期间向头部约束的小鼠提供视觉空间 VR 环境，以研究空间学习行为背后的神经回路。
Protocol
本协议中的所有程序均已获得纽约州精神病学研究所机构动物护理和使用委员会的批准。 注意：单板计算机用于显示与在滚轮上运行头部约束的鼠标相协调的 VR 视觉环境。运动信息以串行输入的形式从 ESP32 微控制器接收，读取耦合到轮轴的旋转编码器。VR环境在Raspberry Pi GPU上使用OpenGL硬件加速渲染，该GPU利用Raspberry Pi的pi3d Python 3D包。然后，渲染环境 通过 投影仪输?…
Representative Results
这种开源的虚拟现实行为设置使我们能够量化舔行为，作为空间学习的读出，因为头部约束的小鼠在虚拟线性轨道环境中导航。将7只4个月大的两性C57BL / 6小鼠置于限制的水时间表上，并首先训练在低水平上连续舔舐，同时在没有VR的情况下在轮子上奔跑以获得随机空间奖励（“随机觅食”）。尽管当以 2 m 随机走廊模式移动到 VR 投影屏幕设置时，它们的性能最初受到影响，但在几次 VR 会话中，?…
Discussion
这款面向鼠标的开源 VR 系统只有在旋转和行为 ESP32 微控制器与单板计算机之间正确建立串行连接（步骤 2）的情况下才能运行（步骤 2），这可以使用 IDE 串行监视器（步骤 2.4.5）进行确认。为了从该协议（步骤4）获得成功的行为结果，必须使小鼠习惯于该装置，并舒适地在轮子上运行以获得液体奖励（步骤4.3-4.5）。这需要足够（但不是过度）的水限制，因为在笼子里随意喝水的小鼠不会…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们要感谢哈维实验室的诺亚·佩蒂特（Noah Pettit）在本手稿中开发协议时的讨论和建议。这项工作得到了BBRF青年研究者奖和NIMH 1R21MH122965（G.F.T.）的支持，此外还有NINDS R56NS128177（R.H.，C.L.）和NIMH R01MH068542（R.H.）。
Materials
| 1/4 " diam aluminum rod | McMaster-Carr | 9062K26 | 3" in length for wheel axle |
| 1/4"-20 cap screws, 3/4" long (x2) | Amazon.com | B09ZNMR41V | for affixing head post holders to optical posts |
| 2"x7" T-slotted aluminum bar (x2) | 8020.net | 1020 | wheel/animal mounting frame |
| 6" diam, 3" wide acrylic cylinder (1/8" thick) | Canal Plastics | 33210090702 | Running wheel (custom width cut at canalplastics.com) |
| 8-32 x 1/2" socket head screws | McMaster-Carr | 92196A194 | fastening head post holder to optical post |
| Adjustable arm (14") | Amazon.com | B087BZGKSL | to hold/adjust lick spout |
| Analysis code (MATLAB) | custom written | file at github.com/GergelyTuri/HallPassVR/software/Analysis code | |
| Axle mounting flange, 1/4" ID | Pololu | 1993 | for mounting wheel to axle |
| Ball bearing (5/8" OD, 1/4" ID, x2) | McMaster-Carr | 57155K324 | for mounting wheel axle to frame |
| Behavior ESP32 code | custom written | file at github.com/GergelyTuri/HallPassVR/software/Arduino code/Behavior board | |
| Black opaque matte acrylic sheets (1/4" thick) | Canal Plastics | 32918353422 | laser cut file at github.com/GergelyTuri/HallPassVR/hardware/VR screen assembly |
| Clear acrylic sheet (1/4" thick) | Canal Plastics | 32920770574 | laser cut file at github.com/GergelyTuri/HallPassVR/hardware/VR wheel assembly |
| ESP32 devKitC v4 (x2) | Amazon.com | B086YS4Z3F | microcontroller for behavior and rotary encoder |
| ESP32 shield | OpenMaze.org | OMwSmall | description at www.openmaze.org (https://claylacefield.wixsite.com/openmazehome/copy-of-om2shield). ZIP gerber files at: https://github.com/claylacefield/OpenMaze/tree/master/OM_PCBs |
| Fasteners and brackets | 8020.net | 4138, 3382,3280 | for wheel frame mounts |
| goniometers | Edmund Optics | 66-526, 66-527 | optional for behavior. Fine tuning head for imaging |
| HallPassVR python code | custom written | file at github.com/GergelyTuri/HallPassVR/software/HallPassVR | |
| Head post holder | custom design | 3D design file at github.com/GergelyTuri/HallPassVR/hardware/VR head mount/Headpost Clamp | |
| LED projector | Texas Instruments | DLPDLCR230NPEVM | or other small LED projector |
| Lick spout | VWR | 20068-638 | (or ~16 G metal hypodermic tubing) |
| M 2.5 x 6 set screws | McMaster-Carr | 92015A097 | securing head post |
| Matte white diffusion paper | Amazon.com | screen material | |
| Metal headposts | custom design | 3D design file at github.com/GergelyTuri/HallPassVR/hardware/VR head mount/head post designs | |
| Miscellenous tubing and tubing adapters (1/16" ID) | for constructing the water line | ||
| Optical breadboard | Thorlabs | as per user's requirements | |
| Optical posts, 1/2" diam (2x) | Thorlabs | TR4 | for head fixation setup |
| Processing code | custom written | file at github.com/GergelyTuri/HallPassVR/software/Processing code | |
| Raspberry Pi 4B | raspberry.com, adafruit.com | Single-board computer for rendering of HallPassVR envir. | |
| Right angle clamp | Thorlabs | RA90 | for head fixation setup |
| Rotary encoder (quadrature, 256 step) | DigiKey | ENS1J-B28-L00256L | to measure wheel rotation |
| Rotary encoder ESP32 code | custom written | file at github.com/GergelyTuri/HallPassVR/software/Arduino code/Rotary encoder | |
| SCIGRIP 10315 acrylic cement | Amazon.com | ||
| Shaft coupler | McMaster-Carr | 9861T426 | to couple rotary encoder shaft with axle |
| Silver mirror acrylic sheets | Canal Plastics | 32913817934 | laser cut file at github.com/GergelyTuri/HallPassVR/hardware/VR screen assembly |
| Solenoid valve | Parker | 003-0137-900 | to administer water rewards |
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Citar este artículo
Lacefield, C., Cai, H., Ho, H., Dias, C., Chung, H., Hen, R., Turi, G. F. An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice. J. Vis. Exp. (193), e64863, doi:10.3791/64863 (2023).