Dataset (NWB format) from Gao et al (2018) A cortico-cerebellar loop for motor planning. Nature, Nov;563(7729):113-116.

<strong>Summary</strong> These experiments measure neuronal responses from anterior lateral motor cortex (ALM) and deep cerebellar nucleus (CN) of adult mice performing pole location discrimination with a short-term memory. In some cases, we manipulate activity of one brain region while recording from the other region. Dataset: <em>Li N (2018). Extracellular recordings from anterior lateral motor cortex (ALM) and cerebellar nucleus neurons of adult mice performing a tactile decision behavior. </em> Data included in this release: 34 sessions (9 mice), ALM recording during fastigial or dentate photoactivation 20 sessions (4 mice), ALM recording during DCN photoinhibition <em>(Data already available at: </em><em>http://dx.doi.org/10.6080/K0NS0S26</em><em>)</em> 185 sessions (18 mice), CN recording. In some sessions, ALM photoinhibition was tested <em>(Data included in this release. Data is in NWB format).</em> Data from the follow publication: <em>Gao Z, Davis C, Thomas AM, Economo MN, Abrego AM, Svoboda K, De Zeeuw CI, Li N (2018). A cortico-cerebellar loop for motor planning. Nature, Nov;563(7729):113-116. doi: 10.1038/s41586-018-0633-x. Epub 2018 Oct 17.</em> <strong>Animals</strong> This dataset contains data from 31 mice (age &gt; P60, both male and female mice, Supplemental Table 1). 9 C57B1/6 mice were used for ALM recordings during photo-activation of the CN. 4 L7-cre (Lewis et al., 2004) crossed to Ai32 (Rosa26-LSL-ChR2-EYFP, JAX Stock#012569) (Madisen et al., 2012) mice were used for ALM recordings during CN photo-inhibition. 10 C57B1/6 mice were used for CN recording experiments. 8 VGAT-ChR2-EYFP mice (Jackson laboratory, JAX Stock#014548) (Zhao et al., 2011) were used for CN recordings during ALM photo-inhibition. <strong>Experimental methods</strong> Detailed experimental methods are described in the manuscript (Gao et al 2018). <strong><em>Behavior</em></strong> Mice measured the location of an object using their whiskers during a sample epoch (1.3 s) (O'Connor et al., 2010). After the sample epoch they must hold their decision about object location in memory for a delay period (1.3 s) (Guo et al., 2014). At the end of the delay period, an auditory cue (0.1) instructed the mice to report their decision with directional licking (“lick left”/”lick right”). <strong><em>CN ChR2 photo-activation </em></strong> For ChR2 photo-activation of the CN, wild-type mice injected with AAV2-hSyn1-(h134R)ChR2-EYFP virus were used. Light from a 473 nm laser (Laser Quantum, Part# Gem 473) was controlled by an acousto-optical modulator (AOM; Quanta Tech) and a shutter (Vincent Associates). To prevent the mice from distinguishing photostimulation trials from control trials using visual cues, a ‘masking flash’ was delivered using 470 nm LEDs (Luxeon Star) near the eyes of the mice. The masking flash began as the pole started to move and continued through the end of the epoch in which photostimulation could occur. The photostimulus was pulses of light (5 ms pulse duration) delivered at 20 Hz and a range of peak powers (5, 10, 15mW). The power values reported in the paper indicate average powers (0.5, 1, 1.5 mW). The powers were measured at the fiber tip. The photostimulus started at the beginning of a task epoch and continued for 0.455 s (10 pulses). <strong><em>CN photo-inhibition</em></strong> In L7-cre × Ai32 mice, ChR2 was expressed in cerebellar Purkinje cells. We photostimulated Purkinje cells to inhibit neurons in the CN. The photostimulus was a 40 Hz sinusoid (average power, 4.5 mW) lasting for 1.3 sec, including a 100-200ms linear ramp during the laser offset to reduce rebound neuronal activity. <strong><em>ALM photo-inhibition</em></strong> ALM is centered on bregma anterior 2.5 mm, lateral 1.5 mm (Chen et al., 2017; Guo et al., 2014; Li et al., 2016). For photo-inhibition of ALM, we photostimulated cortical GABAergic neurons in VGAT-ChR2-EYFP mice (8 mice). Photostimulation was performed through the clear-skull cap implant by directing the blue laser over the skull (beam diameter: 400 µm at 4σ, bregma anterior 2.5 mm, lateral 1.5 mm). The light transmission through the intact skull was 50% (Guo et al., 2014). We photo-inhibited ALM for 1.3 s at the beginning of the delay epoch, including a 100 ms linear ramp at the laser offset to minimize rebound excitation. This photostimulus was empirically determined to produce robust photo-inhibition in ALM (Guo et al., 2014; Li et al., 2016). The photo-inhibition silenced 90% of spikes in a cortical area of 1mm radius (at half-max) through all cortical layers. For unilateral ALM photo-inhibition, we used a 40 Hz sinusoidal photostimulus (1.5mW average power at the skull surface) at 2.5 mm anterior and 1.5 mm lateral from bregma. For bilateral ALM photo-inhibition, we used a constant photostimulus and a scanning galvo (GVSM002, Thorlabs), which stepped the laser beam sequentially through the photo-inhibition sites at the rate of 1 step per 5 ms (step time: 0.2 ms; dwell time: 4.8 ms; measured using a photodiode). 8 photo-inhibition sites were spaced in 1 mm at anterior 2-3 mm and lateral 1-2 mm from bregma, covering ALM. Peak power was adjusted based on the number of photo-inhibition sites to achieve 1.5 mW average power per site. <strong><em>Electrophysiology</em></strong> Extracellular spikes were recorded using 32-channel NeuroNexus silicon probes (Part# A4x8-5mm-100-200-177) or 64-channel Cambridge NeuroTech silicon probes (H2 acute probe, 25 µm spacing, 2 shanks). The 32-channel voltage signals were multiplexed, digitized by a PCI6133 board at 400 kHz (National Instruments) at 14 bit, demultiplexed (sampling at 25,000 Hz) and stored for offline analysis. The 64-channel voltage signals were amplified and digitized on an Intan RHD2164 64-Channel Amplifier Board (Intan Technology) at 16 bit, recorded on an Intan RHD2000-Series Amplifier Evaluation System (sampling at 20,000 Hz) using Open-Source RHD2000 Interface Software from Intan Technology (version 1.5.2), and stored for offline analysis. The extracellular recording traces were band-pass filtered (300-6 kHz). Events that exceeded an amplitude threshold (4 standard deviations of the background) were subjected to manual spike sorting to extract single-units (Guo et al., 2014). <strong>Data analysis</strong> For ALM recordings, units are classified based on spike shape. Spike widths were computed as the trough-to-peak interval in the mean spike waveform. Units with spike width &lt; 0.35 ms were defined as fast-spiking neurons (82/1309) and units with spike widths &gt; 0.45 ms as putative pyramidal neurons (1194/1309). Units with intermediate values (0.35 - 0.45 ms, 33/1309) were excluded from analyses. This classification was previously verified by optogenetic tagging of GABAergic neurons (Guo et al., 2014). For CN recordings, units are classified based on recording location. We estimated unit locations based on recording track labeling, recording depth, and the lamination of activity patterns across the recording shanks. In <em>post-hoc</em> histology, CN boundaries were visible in DAPI staining. <strong>References</strong> Chen, T.W., Li, N., Daie, K., and Svoboda, K. (2017). A Map of Anticipatory Activity in Mouse Motor Cortex. Neuron<em> 94</em>, 866-879 e864. Guo, Z.V., Li, N., Huber, D., Ophir, E., Gutnisky , D.A., Ting, J.T., Feng, G., and Svoboda, K. (2014). Flow of cortical activity underlying a tactile decision in mice. Neuron<em> 81</em>, 179-194. Lewis, P.M., Gritli-Linde, A., Smeyne, R., Kottmann, A., and McMahon, A.P. (2004). Sonic hedgehog signaling is required for expansion of granule neuron precursors and patterning of the mouse cerebellum. Dev Biol<em> 270</em>, 393-410. Li, N., Daie, K., Svoboda, K., and Druckmann, S. (2016). Robust neuronal dynamics in premotor cortex during motor planning. Nature. Madisen, L., Mao, T., Koch, H., Zhuo, J.M., Berenyi, A., Fujisawa, S., Hsu, Y.W., Garcia, A.J., 3rd, Gu, X., Zanella, S.<em>, et al.</em> (2012). A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nature neuroscience<em> 15</em>, 793-802. O'Connor, D.H., Clack, N.G., Huber, D., Komiyama, T., Myers, E.W., and Svoboda, K. (2010). Vibrissa-based object localization in head-fixed mice. The Journal of neuroscience : the official journal of the Society for Neuroscience<em> 30</em>, 1947-1967. Zhao, S., Ting, J.T., Atallah, H.E., Qiu, L., Tan, J., Gloss, B., Augustine, G.J., Deisseroth, K., Luo, M., Graybiel, A.M.<em>, et al.</em> (2011). Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nature methods<em> 8</em>, 745-752.摘要 本系列实验记录了成年小鼠在执行带有短时记忆的杆位辨别任务时，其前外侧运动皮层（anterior lateral motor cortex, ALM）与小脑深部核团（deep cerebellar nucleus, CN）的神经元活动响应。在部分实验中，我们在记录其中一个脑区活动的同时，对另一脑区的神经元活动进行了操控。 数据集来源：Li N (2018). 《成年小鼠执行触觉决策行为时前外侧运动皮层与小脑核团神经元的细胞外记录》。 本发布包含以下数据集： 1. 34个会话记录（9只小鼠）：包括顶核或齿状核光激活期间的ALM记录（20个会话，4只小鼠）；以及DCN光抑制期间的ALM记录【数据已提前发布于：http://dx.doi.org/10.6080/K0NS0S26】； 2. 185个会话记录（18只小鼠）：CN神经元活动记录。 部分会话中测试了ALM光抑制操作【数据包含于本次发布中，数据格式为NWB】。 本数据集还包含来自以下发表论文的相关数据：Gao Z, Davis C, Thomas AM, Economo MN, Abrego AM, Svoboda K, De Zeeuw CI, Li N (2018). 《用于运动规划的皮层-小脑环路》. *Nature*, Nov;563(7729):113-116. doi: 10.1038/s41586-018-0633-x. Epub 2018 Oct 17. ## 实验动物 本数据集涵盖31只小鼠的实验数据（年龄＞P60，雌雄均包含，详细信息见补充表1）。其中： - 9只C57B1/6小鼠用于小脑核团光激活期间的ALM记录； - 4只L7-cre（Lewis等，2004）与Ai32（Rosa26-LSL-ChR2-EYFP，JAX品系编号012569）（Madisen等，2012）杂交的小鼠，用于小脑核团光抑制期间的ALM记录； - 10只C57B1/6小鼠用于小脑核团记录实验； - 8只VGAT-ChR2-EYFP小鼠（杰克逊实验室，JAX品系编号014548）（Zhao等，2011）用于ALM光抑制期间的小脑核团记录。 ## 实验方法 详细的实验方法已在相关论文（Gao等，2018）中完整描述。 ### 行为学范式 小鼠在样本阶段（1.3s）通过胡须感知物体的位置（O'Connor等，2010）。样本阶段结束后，小鼠需要在延迟期（1.3s）内将物体位置的决策判断维持于短时记忆中（Guo等，2014）。延迟期结束时，听觉提示（0.1）指示小鼠通过定向舔舐行为报告其决策结果（“左舔”/“右舔”）。 ### 小脑核团ChR2光激活 针对小脑核团的ChR2光激活实验，我们使用了注射有AAV2-hSyn1-(h134R)ChR2-EYFP病毒的野生型小鼠。473nm激光（Laser Quantum，货号Gem 473）的输出通过声光调制器（acousto-optical modulator, AOM；Quanta Tech）与机械快门（Vincent Associates）进行精准控制。为避免小鼠通过视觉线索区分光刺激试次与对照试次，我们在小鼠眼部附近使用470nm LED（Luxeon Star）施加“遮蔽闪光”：遮蔽闪光在杆状物开始移动时启动，并持续至所有可能施加光刺激的实验阶段结束。光刺激采用频率20Hz、单脉冲时长5ms的脉冲光，峰值功率范围为5、10、15mW。论文中报告的功率值为光纤尖端处测得的平均功率（0.5、1、1.5mW）。光刺激于任务阶段开始时启动，持续时长0.455s（共包含10个脉冲）。 ### 小脑核团光抑制 在L7-cre×Ai32杂交小鼠中，ChR2蛋白特异性表达于小脑浦肯野细胞内。我们通过光刺激浦肯野细胞，实现对小脑核团神经元活动的抑制。光刺激采用持续1.3s的40Hz正弦波光信号（平均功率4.5mW），在激光关闭阶段加入100-200ms的线性斜坡功率衰减，以降低神经元的反弹活动。 ### 前外侧运动皮层光抑制 ALM的解剖定位为前囟前2.5mm、外侧1.5mm处（Chen等，2017；Guo等，2014；Li等，2016）。针对ALM的光抑制实验，我们对VGAT-ChR2-EYFP小鼠（共8只）的皮层GABA能神经元进行光刺激。通过透明颅骨植入物，将蓝色激光对准颅骨表面进行光刺激：光斑直径在4σ标准差处为400μm，刺激坐标为前囟前2.5mm、外侧1.5mm。光线透过完整颅骨的透射率约为50%（Guo等，2014）。我们在延迟阶段开始时对ALM施加1.3s的光抑制，激光关闭阶段加入100ms的线性斜坡功率衰减，以最小化神经元的反弹兴奋。该光刺激方案经实验验证可在ALM中产生稳定可靠的光抑制效果（Guo等，2014；Li等，2016），可使半径1mm的皮层区域（半最大效应处）内90%的动作电位（spike）活动沉默，且该抑制效果覆盖所有皮层层次。 单侧ALM光抑制采用40Hz正弦光刺激（颅骨表面平均功率1.5mW），刺激坐标为前囟前2.5mm、外侧1.5mm处。双侧ALM光抑制则采用连续光刺激搭配扫描振镜（GVSM002，Thorlabs），以每5ms1步的速率依次扫描光刺激位点（步进时间：0.2ms；驻留时间：4.8ms；使用光电二极管进行参数测量）。共设置8个光刺激位点，从前囟前2-3mm、外侧1-2mm处以1mm间距排布，完全覆盖ALM区域。根据光刺激位点的数量调整激光峰值功率，确保每个位点的平均功率为1.5mW。 ### 电生理记录 细胞外动作电位记录采用32通道NeuroNexus硅探针（货号A4x8-5mm-100-200-177）或64通道Cambridge NeuroTech硅探针（H2急性探针，电极间距25μm，包含2个探针臂）。32通道电压信号经多路复用后，由PCI6133数据采集板以400kHz采样率、14位精度进行数字化，随后解多路复用（采样率25,000Hz）并存储以供离线分析。64通道电压信号经Intan RHD2164 64通道放大器板放大并以16位精度数字化，通过Intan RHD2000系列放大器评估系统进行记录（采样率20,000Hz），使用Intan Technology开源的RHD2000接口软件（版本1.5.2）完成数据存储，最终用于离线分析。细胞外记录信号经带通滤波（300-6kHz）处理。当信号振幅超过背景噪声的4倍标准差阈值时，将对该事件进行人工动作电位分选，以提取单一神经元单位（Guo等，2014）。 ## 数据分析 针对ALM记录数据，神经元单位根据动作电位波形特征进行分类。动作电位宽度以平均动作电位波形的峰谷间期计算。将动作电位宽度＜0.35ms的单位定义为快放电神经元（82/1309），动作电位宽度＞0.45ms的单位定义为疑似锥体神经元（1194/1309）。动作电位宽度介于0.35-0.45ms之间的单位（33/1309）被排除在本次数据分析之外。该神经元分类方法此前已通过GABA能神经元的光遗传标记得到验证（Guo等，2014）。 针对CN记录数据，神经元单位根据记录位置进行分类。我们通过记录轨迹标记、记录深度以及记录探针臂跨区域的神经元活动模式分层结构，来估算单个神经元单位的记录位置。在事后组织学染色实验中，通过DAPI染色可清晰观察到小脑核团的边界。 ## 参考文献 1. Chen TW, Li N, Daie K, Svoboda K. (2017). 小鼠运动皮层的预测活动图谱. *Neuron* 94:866-879 e864. 2. Guo ZV, Li N, Huber D, Ophir E, Gutnisky DA, Ting JT, Feng G, Svoboda K. (2014). 小鼠触觉决策过程中皮层活动的流动模式. *Neuron* 81:179-194. 3. Lewis PM, Gritli-Linde A, Smeyne R, Kottmann A, McMahon AP. (2004). Sonic Hedgehog信号通路对小鼠小脑颗粒细胞前体增殖与模式形成的调控作用. *Dev Biol* 270:393-410. 4. Li N, Daie K, Svoboda K, Druckmann S. (2016). 运动规划过程中运动前皮层的稳定神经元动态. *Nature*. 5. Madisen L, Mao T, Koch H, Zhuo JM, Berenyi A, Fujisawa S, Hsu YW, Garcia AJ 3rd, Gu X, Zanella S, et al. (2012). 用于光诱导激活与沉默的Cre依赖型光遗传转基因小鼠工具库. *Nature Neuroscience* 15:793-802. 6. O'Connor DH, Clack NG, Huber D, Komiyama T, Myers EW, Svoboda K. (2010). 头部固定小鼠基于胡须的物体定位行为. *The Journal of Neuroscience : 美国神经科学学会官方期刊* 30:1947-1967. 7. Zhao S, Ting JT, Atallah HE, Qiu L, Tan J, Gloss B, Augustine GJ, Deisseroth K, Luo M, Graybiel AM, et al. (2011). 用于神经环路功能光遗传解析的细胞类型特异性通道视紫红质-2转基因小鼠. *Nature Methods* 8:745-752.