C.M.L. 承认空军科学研究办公室 (FA9550-14-1-0136)、哈佛大学物理科学和工程加速器奖以及全国卫生主任先驱奖 (1DP1EB025835-01). T.G.S. 承认国防部 (DoD) 通过国防科学 & 工程研究生奖学金 (NDSEG) 计划提供的支持。伦理承认美国心脏协会 (16POST27250219) 的奖学金支持, 以及来自国立卫生研究院老龄研究所的独立奖 (父母 K99/R00) 的途径。这项工作是在哈佛大学纳米系统中心 (CNS) 的一部分进行的, 这是国家纳米技术协调基础设施网络 (NNCI) 的成员, 由国家科学基金会根据 NSF ECCS 奖编号支持。1541959。

所有关于脊椎动物的程序都由哈佛大学机构动物护理和使用委员会 (IACUC) 批准。
1. 网格电子的制作
注: 本节描述的程序旨在用于标准的大学洁净室设施, 如哈佛大学纳米系统中心 (CNS)。这一设施以及类似设施可供美国各地的外部用户访问, 例如, 作为国家科学基金会 (NSF) 支持的国家纳米技术基础设施网络 (NNIN) 的一部分。在这些设施中, 本节所述的许多工具、设备和材料都是在进入洁净室设施的同时提供的, 不需要单独购买。
注意: 在制造网格电子学中使用的许多化学物质都是有害的, 包括抵抗、CD-26、卸妆 PG、SU-8 开发人员和镍蚀刻溶液。在使用和实施之前, 请查阅这些化学品的材料安全数据表 (MSDS), 并在任何时候遵守适当的安全措施。
<ol><li>将 100 nm 的镍热蒸发到清洁的硅晶片上。 注: 典型沉积参数为 5 x 10-7 T 的基压和Å/s 的速率。薄层的镍作为牺牲层, 稍后将被解散, 以释放网状电子从晶片。</li>
	<li>使用第一个 pl 掩模 (pl mask-1), 以 SU-8 负光刻胶 (图 2A) 定义网格电子的底层钝化层。 注意: PL 是一种标准微细加工技术, 紫外线 (UV) 光照射在感光基板上的面具上。光要么使不溶性 (阴性抵抗) 或可溶性 (正面抵抗) 暴露的区域在基体上。掩码在计算机辅助设计 (CAD) 软件中绘制, 然后通常从供应商处订购。掩码光刻用于将掩码与基板上的现有图案对齐, 并将其暴露于 UV 光。制造网状电子学需要四种不同的面具 (pl mask-1 通过 pl mask-4)。我们的面具设计是可要求或从资源网站, meshelectronics.org。<ol>
<li>自旋涂层 SU-8 2000.5 负光刻胶到硅片上, 在 4000 rpm 大约 SU-8 厚度的 400–500 nm。</li>
		<li>软烘烤的硅片上的热板上的1分钟在65°c, 其次是1分钟95°c。</li>
		<li>将晶片装入掩模光刻, 以将 SU-8 与底网 SU-8 层对应的 PL mask-1 曝光。暴露在一条 i 线 (365 毫微米波长) 药量 100 mJ/cm2。</li>
		<li>后烘烤硅片在热板上1分钟, 在65°c, 其次是1分钟95°c。</li>
		<li>将晶片浸入 SU-8 开发者的托盘中。轻轻搅动溶液2分钟, 直到 SU-8 中的网格模式得到充分发展。在异丙醇的托盘中冲洗1分钟, 然后吹干。</li>
		<li>硬烤的硅片上的热板在180°c 1 小时。 注: SU-8 通常在150摄氏度和250摄氏度以下发展后硬烤。硬烘烤退火在开发过程中可能形成的任何表面裂纹, 并进一步 crosslinks SU-8 以确保机械稳定性。硬烘烤在180°c 为底部 SU-8 层和190°c 为顶 SU-8 层数为网格电子提供好结果。</li>
	</ol></li>
	<li>使用 PL mask-2 定义金属互连和 i/o 垫 (图 2B)。
	<ol>
<li>自旋涂层 LOR3A 到硅片上 4000 rpm, 大约厚度为 300 nm。 注: LOR3A 是一种以 polydimethylglutarimide 为基础的抗性, 在随后的金属化过程中速度削弱。</li>
		<li>在180摄氏度的热板上烘烤硅片5分钟。</li>
		<li>自旋涂层 S1805 正光刻胶在 4000 rpm 大约厚度为 500 nm。</li>
		<li>在115摄氏度的热板上烘烤硅片1分钟。</li>
		<li>将晶片装入掩模光刻, 以将 S1805 与金属互连和 i/o 垫片对应的 PL mask-2 曝光。暴露在 h 线 (405 毫微米波长) 药量 40 mJ/cm2。</li>
		<li>将晶片浸入 CD-26 光刻胶显影盘中。轻轻搅动溶液1分钟, 直到金属互连线模式得到充分发展。在一托盘的去离子水 (DI) 冲洗1分钟, 吹干。</li>
		<li>热蒸发3毫微米 Cr 跟随80毫微米的 Au。 注: 基压最大为 5 x 10-7 T, 沉积速率为1Å/秒, 通常产生最佳膜质量。</li>
		<li>将晶片浸入一台去除 PG 的扁平烧杯中大约3小时, 直到金属完全切下, 将金属仅留在所需的互连和网格电子的 i/o 垫区。用异丙醇冲洗, 吹干。</li>
	</ol></li>
	<li>使用 PL mask-3 来定义 Pt 电极 (图 2C)。
	<ol>
<li>重复步骤1.3.1 通过1.3.4。</li>
		<li>将晶片装入掩模光刻, 将 S1805 与铂电极对应的 PL mask-3 暴露出来。暴露在 h 线剂量的 40 mJ/cm2。</li>
		<li>将晶片浸入 CD-26 光刻胶显影盘中。轻轻搅动溶液1分钟, 直到铂电极模式得到充分发展。在一托盘中漂洗1分钟, 吹干。</li>
		<li>使用电子束蒸发器沉积 3 nm 的 Cr, 其次是 50 nm 的 Pt。 注: 典型沉积参数为基压为 5 x 10-7 T, 速率为2Å/秒。</li>
		<li>将晶片浸入一只约3小时的卸妆 PG 的扁平烧杯中, 直到金属完全切下, 只在所需的网格电子电极位置留下 Pt。用异丙醇冲洗, 吹干。</li>
	</ol></li>
	<li>使用 PL mask-4 定义 SU-8 负光刻胶 (图 2D) 的网格电子的顶钝化层。
	<ol>
<li>重复步骤1.2.1 通过 1.2.5, 除了暴露与 PL mask-4 对应的顶钝化网层 SU-8。</li>
		<li>硬烤的硅片上的热板在190°c 1 小时。 注: 此温度比底部 SU-8 (步骤 1.2.6) 的硬烘焙高10摄氏度。这种升高的温度略微重排版底部 SU-8, 导致它与顶部 SU-8 层合并, 从而形成一个单一的、连续的 SU-8 结构。</li>
	</ol></li>
	<li>网格电子现在完成 (图 2E和图 3);溶解镍牺牲层, 将其从硅晶片中释放出来。
	<ol>
<li>用 50 W 的氧等离子体处理晶片1分钟。这种氧化 SU-8 表面, 使其亲水性, 并允许在水溶液中容易悬浮的网格。</li>
		<li>在扁平烧杯中, 将盐酸、氯化铁和 DI 的容积率分别为 1:1:20, 以此来解决镍蚀刻。</li>
		<li>将晶片浸入镍蚀刻溶液的扁烧杯中大约3小时, 直到网格电子完全从硅晶片中释放。</li>
		<li>使用巴斯德吸管将释放的网状电子探针从 Ni 蚀刻转移到100毫升的 DI 烧杯中。将网格电子传送到 DI 的新鲜烧杯中至少3次, 以确保漂洗。</li>
		<li>使用巴斯德吸管将网格电子转移到 70%/30% 乙醇/水溶液中进行消毒, 然后使用吸管将网格电子转移到无菌水中冲洗。 注: 灭菌后, 网格电子可以转移到其他解决方案, 以功能化。例如, 为了促进细胞黏附, 网状电子可以转移到一个水溶液的聚 d-赖氨酸 (1 毫克/毫升) 为24小时。</li>
		<li>使用巴斯德吸管将网状电子探针转移到100毫升的无菌1x 磷酸盐缓冲盐水 (PBS)在体内注射之前。</li>
	</ol></li>
</ol>2. 将网格电子装入针头
<ol><li>吸管持有人购买的是开放的流动两端。密封的两端环螺钉紧固件与环氧树脂, 所以在注射过程中没有泄漏 (图 4A)。在继续之前, 让环氧树脂硬化。</li>
	<li>将玻璃毛细管针插入吸管支架。使用圆形拧紧螺钉和圆锥垫圈将其固定到位 (图 4B)。本协议采用400µm 内径 (650 µm 外径) 玻璃毛细管针。其他毛细针材料(例如, 金属) 和直径可以使用, 但可能需要改变网格电子学的设计, 以确保可注射性。 注: 我们实验室使用了从150µm 到1.17 毫米内径 (250 µm 到1.5 毫米外径) 的毛细管针。通过将横向和纵向 SU-8 网格元素之间的相交角更尖锐, 减小 SU-8 网格元素的宽度, 使用较薄的 SU-8, 使用较粗的 (i. e。较大的单元单元) 网格结构。光掩膜为更小的毛细管针设计的网格是可利用的请求或从资源站点, meshelectronics.org。玻璃毛细管针的内径为400µm 和外径550µm 也可在商业上。这些减少了注射引起的急性组织损伤, 同时保持了与需要400µm 内径的网格的兼容性, 但它们并没有用于此处描述的研究。</li>
	<li>使用2–4厘米长的毛细管管, 将1毫升注射器连接到吸管托架的侧出口。</li>
	<li>将玻璃毛细管针插入包含网格电子的 PBS 的100毫升烧杯中。将针的末端定位在网格电子探针的 i/o 垫附近, 并手动收回注射器, 以便在针上绘制网状电子探针 (图 5和辅助视频 1)。 注: 当网格电子探头悬浮在溶液中时, 用肉眼可以很容易地识别出 i/o 垫片、阀杆互连区域和网格设备区域。这样就可以明确地将网格电子加载到针中, 并且方向正确。</li>
	<li>推/拉注射器, 而仍然浸泡在生理盐水, 以调整网格电子在针的位置。 注意: 理想定位是与 ultraflexible 网格设备区域尽可能接近针的末端。这种配置最大限度地减少了将注入大脑的液体体积, 同时保证了设备区域首先被注入, 并且 i/o 垫最后。</li>
	<li>小心地从吸管持有者的侧出口处分离毛细管管。慢慢地将其分离, 以避免产生能改变针内网状电子位置的吸力。</li>
</ol>3. 将网状电子定向到活鼠脑中
注: 小鼠用腹腔注射麻醉, 混合了75毫克/千克氯胺酮和1毫克/千克 dexdomitor。术前采用趾夹法对麻醉程度进行了验证。在麻醉下, 在37摄氏度恒温毯上放置鼠标, 保持体温。为手术实施了适当的不育技术, 包括但不限于热处理所有金属手术器械, 使用前1小时, 使用消毒手套, 使用热珠灭菌器贯穿整个手术, 维持无菌领域在手术现场周围, 用70% 乙醇消毒塑料器械, 脱毛头皮皮肤在切口前用碘伏准备。为生存手术, 在手术结束后, antiobiotic 软膏被应用在伤口周围, 老鼠被送回一个装有37摄氏度加热垫的笼子里。老鼠没有被留在无人看管, 直到他们恢复了足够的意识, 以维持胸骨卧床。在手术后, 小鼠通过腹腔注射0.05 毫克/千克体重每12小时剂量的丁丙诺啡镇痛。小鼠在手术后与其他动物隔绝。通过腹腔注射戊巴比妥的剂量为270毫克/千克体重或通过 transcardial 灌注对小鼠进行安乐死 (见步骤 6.1)。调查人员可以指盖革,等等。30, 科比,等等。31, 和量具,等等。32有关啮齿动物立体定向手术的详细信息。
<ol><li>麻醉鼠标并将其固定在立体定向的框架中。</li>
	<li>将眼部润滑剂应用于鼠标的眼睛, 以防止麻醉时的干燥。</li>
	<li>使用牙钻和立体定向的框架打开一个开颅手术, 在所需的头骨坐标。打开第二个开颅手术远离注射部位插入不锈钢接地螺钉或电线。</li>
	<li>用牙科水泥把一个夹紧的基底固定在头骨上。在基板上切下大约1毫米宽的间隙, 以提高过程后期折叠步骤的可靠性。 注意: 一个扁平的柔性电缆 (ZIF) 切割到 "L" 形状工作良好 (图 7A), 虽然许多材料将工作, 只要他们是正确的厚度为32通道零插入力 () 连接器 (设计为0.18 英寸0.05 毫米厚电缆)。</li>
	<li>使用直角端钳将吸管支架与含有网状电子的针安装在立体定向架上 (图 4C和图 6A)。</li>
	<li>使用大约0.5–1米长的毛细管管, 将吸管支架的侧出口连接到注射器泵中固定的5毫升注射器 (图 6D)。 注: 确保毛细管管中没有气泡, 然后再将其连接到吸管架上。气泡可以中断注入过程中的流动, 防止网格电子的平滑、可控交付。</li>
	<li>使用立体定向的框架将针的尖端定位在大脑中所需的起始位置。 注: 这里使用的网格电子探头设计的记录电极分布在长约2毫米和与第一电极位于约0.5 毫米从网格电子的开始边缘 (左-最边缘在图 3A)。因此, 应选择立体定向的坐标, 使起始位置比感兴趣的脑区深0.5 毫米。在掩模设计过程中, 记录电极的分布和位置可以自由选择, 因此, 当记录电极沿其立体定向注入时, 它们会跨越感兴趣的大脑区域。轨迹。</li>
	<li>定位相机 (图 6B), 以显示在玻璃针内的网格电子探头的顶端。一些软件允许用户在屏幕上画一条线来标记网格电子的原始位置。</li>
	<li>通过将注射器泵设置为低速并按下启动来启动流量。10毫升/小时是一个典型的起始流速为400µm 内径毛细管针。如果网格电子探头不在针内移动, 则缓慢增加流量。 注意: 重要的是要尽量减少注入大脑的液体体积, 因为这会损害注射部位周围的组织。最好的结果是, 注射量少于25µL 每1毫米注入网格长度。理想值小于该体积的一半;在我们的实验室, 我们通常注射10–50µL 每4毫米注入网格长度。</li>
	<li>当网格电子探针开始在针内移动时, 用网格电子探针的标记原位置作为参考, 使用立体定位框架以相同的速率收回针。 注: 此过程称为视场 (FoV) 注射方法25, 允许精确地将网格电子传送到目标大脑区域, 而不皱或错位。当网状电子探针开始在针内移动时, 流量往往会降低。在我们的实验室中, 经常需要20–30毫升/小时的流量来克服网格和毛细针壁之间的静态摩擦, 但一旦启动注射过程, 速率就会降低到10毫升/小时。流量和注入量通常较小的小直径毛细管针。</li>
	<li>继续流动的盐水和缩回针, 直到针已经退出头骨。停止注射器泵的流量。</li>
</ol>4. 输入/输出接口
注意: 此时, 网格电子探针已经从大脑中所需的起始点注入, 沿着所选的轨迹。针已被收回, 只是在开颅手术与网状电子互联跨越从大脑到针和 i/o 垫仍然在针 (图 7B)。本节使用印制电路板 (PCB;图 7,图 8) 接口到网格电子探针。PCB 通过绝缘基板将 ZIF 连接器连接到32通道标准放大器接头, 成为神经记录实验的头部阶段。PCB 是可定制的, 以适应各种头部阶段的配置。我们的设计文件可根据要求或资源网站 meshelectronics.org 提供, 可用于从 PCB 制造和组装服务供应商那里廉价购买多氯联苯。
<ol><li>使用立体定向的框架, 仔细引导针, 以使夹紧基板和跨越的差距, 流动的解决方案与注射器泵产生松弛的网状电子互联 (图 7C)。</li>
	<li>一旦针在夹紧衬底上并穿过缝隙, 恢复流速以快速速度将网格电子 i/o 垫片弹出到夹紧衬底上 (图 7D)。</li>
	<li>使用镊子和 DI 吸管, 将 i/o 垫片弯曲至90°角, 尽可能接近第一个 i/o 垫。 注: 弯曲是必要的, 以允许垫在 ZIF 连接器上的 PCB 在随后的步骤。ZIF 连接器与网格电子探头的 32 i/o 垫片的宽度完全相同, 因此, 一个不完美的90°弯曲, 或在第一个 i/o 垫之前没有发生的弯曲, 将导致不得不切断 i/o 垫 (最左边的垫在图 7E)。</li>
	<li>一旦 i/o 垫对齐, 展开, 并在一个90°的角度到网格茎, 干燥他们在原地轻轻流动压缩空气。 注: 不到32个通道的网格电子探头可以与相同的32通道接口板连接。例如, 我们的实验室通常使用16通道的网状电子探头和32通道的多氯联苯。这在 ZIF 连接器中提供了额外的空间, 使得接口更容易, 并且附加的与世隔绝通道通过在记录会话期间的阻抗测试很容易被识别为开路电路。</li>
	<li>从 i/o 垫的边缘将夹紧衬底从大约 0.5–1 mm 的直边切开。还可以切断夹紧衬底的多余部分, 这将阻碍插入 PCB 安装的32通道 ZIF 连接器 (图 7F)。</li>
	<li>将 i/o 垫片插入 PCB 上的 ZIF 接头并关闭闩锁 (图 7G)。使用测量电子测量通道和地面螺钉之间的阻抗, 以确认成功的接口。如果阻抗值太高, 松开 ZIF 连接器, 调整插入, 然后重新测试, 直到确认成功的连接。</li>
	<li>覆盖 ZIF 连接器和暴露的网状电子互联与牙科水泥保护。在承印物的缝隙上翻转 pcb, 用水泥将 pcb 固定在老鼠头骨上 (图 7H)。 注: 弯曲的缺口在间隙减少的机械应变, 有时可以打破网状电子互联。</li>
	<li>允许水泥硬化, 使 PCB 成为一个健壮的, 紧凑的头阶段, 以便在随后的记录会话中进行接口 (图 7I)。</li>
</ol>5. 神经记录实验
<ol><li>将鼠标放在 tailveiner 或其他抑制剂33中。将前置放大器 pcb 插入头级 pcb 上的标准放大器接头。使用单独的电缆接地参考螺钉。</li>
	<li>对于受限制的录制, 请将鼠标放在抑制剂中。使用数据采集系统记录所需时间段内的数据 (图 8A)。</li>
	<li>对于自由移动录音, 在插入前置放大器 PCB 和接地参考螺钉后, 从抑制剂中松开鼠标。在鼠标自由行为时使用数据采集系统记录所需的时间长度 (图 8B)。</li>
	<li>在录制会话结束时, 如果需要, 将鼠标放回抑制剂。卸下接地线和前置放大器, 然后将鼠标放回其笼子并返回到动物设施, 直到下一次录制会话。</li>
</ol>6. 组织学切片、染色和成像
<ol><li>等到所需的时间后注射, 然后麻醉鼠标和 transcardially 灌注与甲醛。将大脑移除、冷冻和 cryosection 10 µm 厚切片。Evilsizor、 et、cryosectioning 的免疫组化和脑组织功能的详细协议。34。 注: 含有网状电子的脑组织可以被固定和切片, 即使监控电子设备留在里面。这是一个独特的能力相比, 传统的神经探针, 必须在切片前删除, 因此可以修改组织或使其难以分析探针-组织界面。</li>
	<li>在 1x PBS 中冲洗冰冻脑组织切片3次。</li>
	<li>在 1x PBS 中, 在0.3% 条海卫 X-100 和5% 山羊血清的溶液中阻断切片。让我们坐在室温下1小时。</li>
	<li>用原抗体的溶液孵育切片。这里使用的主要抗体解决方案是兔抗 NeuN (1:200 稀释), 小鼠抗丝 (1:400 稀释), 和鼠抗胶质细胞 (1:500 稀释) 与0.3% 海卫 X-100 和3% 山羊血清。孵化过夜在4摄氏度。</li>
	<li>用 1x PBS 冲洗部分9次, 共40分钟。</li>
	<li>用二次抗体的溶液孵化大脑部分。在这里使用的二次抗体解决方案是 alexa 氟488山羊抗兔 (1:200 稀释), alexa 氟568山羊抗鼠 (1:200 稀释), 和 Alexa 氟647山羊抗鼠 (1:200 稀释)。在室温下孵育1小时的切片。</li>
	<li>用 1x PBS 冲洗部分9次, 共30分钟。</li>
	<li>使用 antifade mountant 将玻璃幻灯片上的部分装入盖玻片。在成像前将幻灯片放在黑暗中至少24小时。</li>
	<li>用共焦显微镜将 488 nm、561 nm 和 633 nm 激光作为其激发源, 分别为 alexa 氟488、alexa 氟568和 alexa 氟647。利用差分干涉对比 (DIC) 在同一显微镜下对网格电子学进行图像和分析后叠加。</li>
</ol>

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作者没有什么可透露的。

所有的步骤在制造和使用网格电子是重要的, 但一些是特别重要的。在从晶片中释放网格电子之前, 必须氧化表面, 使网格在水溶液中容易悬浮 (步 1.6.1)。如果跳过这一步, 网格通常漂浮在水面上, 使它们难以加载到针头中, 如果它们可以加载, 它们经常粘在玻璃针的两侧, 需要大量的 (> 100 µL) 注入。因此, 在释放前不能氧化表面, 通常意味着不能使用网格, 必须从头开始重新进行制造。另一个关键步骤是弯曲网格电子 "茎" 到 ~ 90°在 i/o 接口 (步骤 4.3)。如果角度小于 90°, 则所有 32 i/o 垫片将不适合 ZIF 连接器;有些将不得不切断结束, 以允许插入, 减少连接电极的数量。这个过程也必须轻轻地做, 以防止茎断裂。
网格电子学的设计可以通过修改光掩膜和使用图 2中概述的相同的制造过程来为各种应用定制。例如, 当用于记录图 9中数据的网格电子探头设计为有32个记录电极横跨老鼠海马和原体感皮层时, ultraflexible 网格中的电极放置可以选择的目标几乎任何大脑区域, 或更大的刺激电极可以被纳入27。保留了相同的基本网格结构和制作工艺, 但对电极的布置和设计进行了调整, 以满足研究的需要。然而, 调查人员应该谨慎使用, 并且总是测试修改过的设计可以通过预定的针头轻易地注入。对网格电子学弯曲力学的小改动对可注射性有很大的影响。一个这样的例子是, 在横向和纵向 SU-8 丝带之间的45°角产生一个网状电子探针, 可以弃注入, 但90°的角度导致一个揉和堵塞针21。
测量记录电极的阻抗有助于故障排除。20-µm 直径圆 Pt 电极应具有 1 MΩ的阻抗级, 当测量频率为1赫体内或 1x PBS29。阻抗明显大于这意味着电极没有暴露, 如可能发生, 如果它是受光刻胶残留, 或没有电连接。例如, 如果在 PL 中的照片掩码上有灰尘导致在 Au 互连中断开连接, 或者在 i/o 接口期间 ZIF 连接器针脚没有联系到其中一个网格 i/o 垫片, 则可能发生后者。阻抗的幅度大约是预期值的一半, 这表明信道可能会短路到相邻的一个, 从而产生两个电极阻抗并联的电路。测量阻抗值在故障排除过程中充当指导;结合网格电子探针的光学显微术, 在下一次制造运行或 i/o 接口尝试时, 通常可以对问题的来源进行相应的识别和修正。
用于急性研究的注射器注射用网状电子学有限, 因为单单位的扣球活动通常没有观察到1周后注射27, 虽然最近的工作 (未发表) 表明, 这个问题是容易克服。观察峰值活动所需时间的关键决定因素是网格设计、随网格电子注入大脑的流体体积以及注射针头的直径, 因为这些影响了组织损伤的程度。注射和愈合率。如果在镍蚀刻释放之前不使用氧等离子体处理网状电子, 则可能需要大量的注入量;即, 如果网格不是亲水性的, 它可以坚持玻璃针。有时, 网格有缺陷, 导致弯曲力学, 使他们难以注入。在网格电子的加载过程中, 重要的是检查网格在针内的移动是否容易且平滑 (如辅助视频 1所示)。如果没有, 则应使用不同的网格电子探针。最好的结果, 无缝的神经接口将实现与理想的注入量10–50µL 每4毫米注入网格长度。最近的结果与更细的网状电子探针注入和/或更小直径毛细管针 (小至150µm 内径, 250 µm 外径) 表明, 单单位扣球可以观察后不久注射 (急性测量)通过更长的时间。这些较细的网格结构的掩码设计文件可通过请求或资源网站 meshelectronics.org 提供。我们估计, 使用400µm 内径 (650 µm 外径) 针, 我们的体内网注射程序的总收益率约为 70%, 虽然产量更接近80–90% 为我们最近的工作与150µm 内径 (250 µm 外径针.最常见的失败原因是 (1) 网格不顺利注入, 导致脑水肿, 从意外的大注射量到大脑, (2) 网损在手动操作时需要在 i/o 接口程序, 和 (3)在注射过程中损害血管的流血。在注射过程中损害血管是罕见的 (原因不到10% 的失败), 并可以进一步减少使用图像引导手术。我们还注意到, 血管损伤是所有涉及脑组织渗透的程序的常见限制, 包括注射病毒微粒进行转染, 植入刚性脑探头, 以及注入网状电子学。
网格电子探头能够稳定地记录和跟踪相同的单个神经元在至少几个月到一年的时间刻度, 并唤起几乎没有慢性免疫应答, 如图 9和图 10所示。与常规深度电极相比, 这是一个显著的优势, 在长期记录实验14中, 通常会减少穗振幅、不稳定信号和慢性炎症,15. 此外, 网格电子学的优点是, 在组织学切片、染色和成像过程中, 它们可以留在组织中, 与传统的探针形成对比, 因为它们过于僵硬, 因此必须在组织学前移除。分析。因此, 网状电子学允许使用免疫组化分析的独特能力, 精确地研究每个记录站点周围的细胞环境。
这里提出的协议开启了令人振奋的新的神经科学机会。微创分娩法和网状电子与脑组织的无缝集成最大限度地减少了神经回路的中断, 避免了慢性免疫应答, 这将有益于大多数类型的慢性神经记录实验。网格电子在长时间内记录和跟踪同一单个神经元的能力将特别感兴趣的是研究人员试图将毫秒级的峰值活动与月-长的过程 (如衰老) 关联起来,脑部疾病的发病机制, 或大脑发育16,18。此外, 还存在扩展和自定义此协议的大量机会, 例如在 PCB 头阶段添加活动电子, 以实现诸如数字复用8、35、无线等功能。通信35,36,37, 信号处理35, 共注入干细胞或聚合物与网状电子, 以帮助组织再生18,38, 39、将纳米线场效应晶体管 (NW FETs) 并入网格电子学中, 用于高度本地化和多功能的脑探头24、29、40、41 ,42。

对大脑进行单神经元分辨率定位的工具的开发对于神经科学和神经学是至关重要的。无创技术的神经研究, 如脑电图 (EEG), 脑 (梅格) 和功能磁共振成像 (fMRI) 已证明有价值的关联大脑活动与行为在人类1, 2, 但它们缺乏研究神经网络在其基本千分尺和毫秒尺度上的结构和动力学所必需的时空分辨率, 分别为3、4。某些皮层脑电图 (ECoG) 探针和光学成像方法使用电压敏感染料成功地记录了在体内5,6的单单位扣球活动, 但它们通常是有效的, 只有接近脑表面, 限制对浅部脑区研究的适用性。相比之下, 植入式电探针可以测量从几乎任何大脑区域自由移动动物的单神经元电生理学, 而不需要荧光标记, 这使得它们对系统级神经科学必不可少, 尤其是从半导体行业的微细加工技术已经把渠道数量推入成百上千的3、7、8、9。凭借这些能力, 植入式电探针对神经科学和神经学作出了许多重要贡献, 包括在视觉系统10中信息处理的基础研究, 神经系统的治疗疾病, 如帕金森氏病11, 和示范脑机接口 (体重指数) 为先进的假肢12,13。
然而, 长期不稳定表现为减少穗振幅和不稳定信号的时间刻度为14,15限制了植入探针的适用性研究相对短期现象, 留下的问题, 如大脑老化和发展基本上没有答案。长期不稳定的局限性是传统探针与脑组织在大小、力学和拓扑14、15、16、17、18之间不匹配的结果。就大小而言, 神经元突触和胞体的直径约为19微米, 而在硅微电极阵列 > 4 倍的情况下, 传统探针通常会显著增大。单个神经元细胞体的大小7,8。这些探针的尺寸相对较大, 可能会扰乱致密神经组织的自然结构和连通性, 从而导致慢性免疫应答, 扰动神经回路的研究。就机械性能而言, 传统探针比植入的极软神经组织要剧烈得多;即使是由10–20µm 厚片的聚酰亚胺制成的 "柔性" 探头, 也比脑组织20、21的硬度至少10万倍。这种不匹配的弯曲刚度导致探针和脑组织之间的相对剪切运动, 导致不可靠的单单位跟踪在延长记录和诱导慢性胶质在植入部位。最后, 常规脑探针的拓扑结构必然排除组织的固体体积。这种拓扑不匹配扰乱了神经回路的连通性, 排除了三维 (3D) 神经元、胶质细胞和脑组织内血管的自然互穿分布, 阻碍了3D 的运输信号分子23。这些传统探针的缺点使它们无法与临床应用和单神经元水平的纵向神经科学研究的长期相容性相结合。
为了克服这些缺点, 我们试图通过开发一种新的 "组织式" 神经探针 (称为网状电子学16、21、24) 来模糊神经和电子系统之间的界线。网格电子解决上述大小, 力学和拓扑的匹配问题, 通过纳入 (1) 结构特征的相同纳米到微米大小的神经组织, (2) 机械性质类似的脑组织, (3) 3D大孔拓扑, 是 > 90% 开放空间, 从而容纳渗透的神经元和扩散分子通过细胞外环境。网状电子探针可以通过注射器和针精确地传送到特定的脑区, 造成最小的急性损伤, 即使在深部脑区21,25。神经元体细胞和轴突已经显示相互渗透开放的3D 网格电子探针结构在几周后注入, 从而创造一个无缝, 无胶质的接口记录电子和周围的脑组织21,26,27. 这些独特的特性使网状电子探针能够稳定地跟踪来自同一单个神经元的峰值活动, 至少一年时间刻度27。此外, 基于光刻技术 (PL) 的网格电子学的制作提供了可合并的电极数量的高可伸缩性, 通过简单接触掩模光刻, 每探头显示的通道计数高达128个电极。28和一个即插即用输入/输出 (i/o) 设计, 允许快速电气连接到外围电子学没有专门设备29。
广泛的研究可能会受益于将网格电子学纳入测量协议。大多数 intracortical 记录实验可以从网状电子学的微创植入过程中受益, 通过注射器注射, 大大降低了植入后的免疫反应, 并有能力离开网格电子在组织在随后的组织学和染色, 以精确分析周围的生物环境的每个记录网站。特别是慢性记录实验将从网状电子的独特能力中获得价值, 以追踪大量的单个神经元数月到数年。这种能力为那些以前不切实际的单神经元分辨率的研究创造了机会, 例如神经回路的纵向老化研究, 发育中的大脑的调查, 以及对性脑病16。
在本协议中, 我们描述了典型的小鼠神经记录实验中使用注射器注射网格电子学的所有关键步骤 (见图 1)。所述步骤包括在许多大学可能在标准的基于 PL 的过程中制造网状电子学, 将网格电子装入标准毛细管针,在体内对网状电子进行立体定向注射, 连接对标准仪器接口进行网格 i/o, 限制或自由移动记录会话, 以及包含网状电子的脑组织的组织学切片。一些研究人员使用网格电子学仅用于组织学研究, 可能不需要电子接口和录音, 在这种情况下, 他们可能跳过这些步骤。在熟悉了这个协议之后, 调查人员应该具备在自己的实验中使用网格电子学所需的所有知识。

<table><tbody><tr><td>Motorized stereotaxic frame</td><td>World Precision Instruments</td><td>MTM-3</td><td>For mouse stereotaxic surgery</td></tr><tr><td>512-channel recording controller</td><td>Intan Technologies</td><td>C3004</td><td>A component of the neural recording system</td></tr><tr><td>RHD2132 amplifier board</td><td>Intan Technologies</td><td>C3314</td><td>A component of the neural recording system</td></tr><tr><td>RHD2000 3 feet ultra thin SPI interface cable</td><td>Intan Technologies</td><td>C3213</td><td>A component of the neural recording system</td></tr><tr><td>Mouse restrainer</td><td>Braintree Scientific</td><td>TV-150&amp;nbsp;STD</td><td>Standard 1.25 inch inner diameter; used to restrain the mouse during restrained recording sessions.</td></tr><tr><td>Si wafers</td><td>Nova Electronic Materials</td><td></td><td>3&quot; P &amp;lt;100&gt; .001-.005 ohm-cm 356-406 &amp;mu;m Thick&lt;br/&gt; Prime Grade SSP Si wafers w/2 Semi-Std. Flats &amp;amp;&lt;br/&gt; 6,000 A&amp;deg;&amp;plusmn;5% Wet Thermal Oxide on both sides.</td></tr><tr><td>Photomasks (chrome on soda lime glass)</td><td>Advance Reproductions</td><td></td><td>Advance Reproductions and other vendors manufacture photomasks from provided design files. Our photomask design files are available by request or from the resource website, meshelectronics.org. Alternatively, some university clean rooms have mask writers for making photomasks on site.</td></tr><tr><td>AutoCAD software</td><td>Autodesk Inc.</td><td></td><td>Design software for drawing photomasks. A free alternative is LayoutEditor. Our photomask design files are available by request or from the resource website, meshelectronics.org.</td></tr><tr><td>Thermal evaporator</td><td>Sharon Vacuum</td><td></td><td>Used to evaporate Ni, Cr, and Au onto mesh electronics during fabrication. Many university clean rooms have this or a similar tool.</td></tr><tr><td>SU-8 2000.5 negative photoresist</td><td>MicroChem Corp.</td><td></td><td>Negative photoresist used to define the bottom and top passivating layers of mesh electronics.</td></tr><tr><td>MA6 mask aligner</td><td>Karl Suss Microtec AG</td><td></td><td>Used to align each photomask to the pattern on the wafer and expose the wafer to UV light. Most university clean rooms have this or a similar tool.</td></tr><tr><td>SU-8 developer</td><td>MicroChem Corp.</td><td></td><td>Used to develop SU-8 negative photoresist following exposure to UV light.</td></tr><tr><td>LOR3A lift-off resist</td><td>MicroChem Corp.</td><td></td><td>Used with Shipley 1805 photoresist to promote undercutting during metal lift-off processes</td></tr><tr><td>Shipley 1805 positive photoresist</td><td>Microposit, The Dow Chemical Company</td><td></td><td>Positive photoresist used to define metal interconnects and Pt electrodes in mesh electronics</td></tr><tr><td>MF-CD-26 positive photoresist developer</td><td>Microposit, The Dow Chemical Company</td><td></td><td>To develop S1805 positive photoresist after exposure in a mask aligner. Many university clean rooms stock this chemical.</td></tr><tr><td>Spin coater</td><td>Reynolds Tech</td><td></td><td>For coating wafers with positive and negative resists. Most university clean rooms have spin coaters.</td></tr><tr><td>PJ plasma surface treatment system</td><td>AST Products, Inc.</td><td></td><td>Used to oxidize the surface of mesh electronics prior to release into aqueous solution. Most university clean rooms have this or a similar tool.</td></tr><tr><td>Electron beam evaporator</td><td>Denton Vacuum</td><td></td><td>For evaporating Cr and Pt during fabrication of mesh electronics. Many university clean rooms have this or a similar tool.</td></tr><tr><td>Remover PG</td><td>MicroChem Corp.</td><td></td><td>Used to dissolve LOR3A and Shipley S1805 resists during metal lift-off</td></tr><tr><td>Ferric chloride solution</td><td>MG Chemicals</td><td>415-1L</td><td>A component of Ni etching solution</td></tr><tr><td>36% hydrochloric acid solution</td><td>Kanto Corp.</td><td></td><td>A component of Ni etching solution</td></tr><tr><td>Glass capillary needles</td><td>Drummond Scientific Co.</td><td></td><td>Inner diameter 0.40 mm, outer diameter 0.65 mm. Other diameters are available.</td></tr><tr><td>Micropipette holder U-type</td><td>Molecular Devices, LLC</td><td>1-HL-U</td><td>Used to hold the glass capillary needles during stereotaxic injection</td></tr><tr><td>1 mL syringe</td><td>NORM-JECT&amp;reg;, Henke Sass Wolf</td><td></td><td>Used for manual loading of mesh electronics into capillary needles</td></tr><tr><td>Polyethylene intrademic catheter tubing</td><td>Becton Dickinson and Company</td><td></td><td>Inner diameter 1.19 mm, outer diameter 1.70 mm</td></tr><tr><td>5 mL syringe</td><td>Becton Dickinson and Company</td><td></td><td>Used in the syringe pump for injection of mesh electronics in vivo</td></tr><tr><td>Eyepiece camera</td><td>Thorlabs Inc.</td><td>DCC1240C</td><td>Used to view mesh electronics within capillary needles during injection</td></tr><tr><td>ThorCam uc480 image acquisition software for USB cameras</td><td>Thorlabs Inc.</td><td></td><td>Used to view mesh electronics within capillary needles during injection</td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>PHD 2000</td><td>Used to flow precise volumes of solution through capillary needles during injection of mesh electronics</td></tr><tr><td>EXL-M40 dental drill</td><td>Osada</td><td>3144-830</td><td>For drilling the craniotomy</td></tr><tr><td>0.9 mm drill burr</td><td>Fine Science Tools</td><td>19007-09</td><td>For drilling the craniotomy</td></tr><tr><td>Hot bead sterilizer 14 cm</td><td>Fine Science Tools</td><td>18000-50</td><td>Used to sterlize surgical instruments</td></tr><tr><td>CM1950 cryosectioning instrument</td><td>Leica Microsystems</td><td></td><td>Used to slice frozen tissue into sections. Many universities have this or a similar tool available in a shared facility.</td></tr><tr><td>0.3% Triton x-100</td><td>Life Technologies</td><td></td><td>Used for histology</td></tr><tr><td>5% goat serum</td><td>Life Technologies</td><td></td><td>Used for histology</td></tr><tr><td>3% goat serum</td><td>Life Technologies</td><td></td><td>Used for histology</td></tr><tr><td>Rabbit anti-NeuN</td><td>Abcam</td><td>ab177487</td><td>Used for histology</td></tr><tr><td>Mouse anti-Neurofilament</td><td>Abcam</td><td>ab8135</td><td>Used for histology</td></tr><tr><td>Rat anti-GFAP</td><td>Thermo Fisher Scientific Inc.</td><td>PA516291</td><td>Used for histology</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Thermo Fisher Scientific Inc.</td><td>P36930</td><td>Used for histology</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich Corp.</td><td>P6407-5MG</td><td>Molecular weight = 70-150 kDA</td></tr><tr><td>Right-angle end clamp</td><td>Thorlabs Inc.</td><td>RA180/M</td><td>Used to attach the pipette holder to the stereotaxic frame</td></tr><tr><td>Printed circuit board (PCB)</td><td>Advanced Circuits</td><td></td><td>Used to interface between mesh electronics and peripheral measurement electronics such as the Intan recording system. Advanced Circuits and other vendors manufacture and assemble PCBs based on provided design files. Our PCB design files are available by request or at the resource site meshelectronics.org</td></tr><tr><td>32-channel standard amplifier connector</td><td>Omnetics Connector Corp.</td><td>A79024-001</td><td>Component assembled onto the PCB</td></tr><tr><td>32-channel flat flexible cable (FFC)</td><td>Molex, LLC</td><td>152660339</td><td>Used as a clamping substrate when interfacing to mesh electronics I/O pads with the PCB-mounted ZIF connector</td></tr><tr><td>32-channel zero insertion force (ZIF) connector</td><td>Hirose Electric Co., LTD</td><td>FH12A-32S-0.5SH(55)</td><td>Component assembled onto the PCB</td></tr></tbody></table>

syringe-injectable mesh electronics for stable chronic rodent electrophysiology

植入性脑电生理探针是神经科学的宝贵工具, 因为它们能够记录来自浅和深脑区的高时空分辨率的神经活动。然而, 由于探针和脑组织之间的机械和结构不匹配, 它们的使用受到阻碍, 通常导致微动和胶质, 导致慢性记录实验中的信号不稳定。相比之下, ultraflexible 网格电子通过注射器注射植入后, 网状探针就形成了一个无缝的、无胶质的界面, 与周围的脑组织, 使单个神经元的稳定跟踪至少一年时间表。本协议详细介绍了在典型的鼠标神经记录实验中使用注射器注射网格电子学的关键步骤, 包括在许多大学可能在标准的基于光刻的过程中制造网状电子学, 加载网状电子成标准毛细管针, 立体定向注射在体内, 连接的网输入/输出到标准仪器接口, 克制或自由移动记录会话, 和组织学切片的大脑含有网状电子的组织。提出了具有代表性的神经记录和组织学数据。熟悉此协议的调查人员将有必要将网格电子学纳入自己的实验, 利用长期稳定的神经接口所提供的独特机会, 如衰老研究过程, 大脑发育和脑部疾病的发病机制。

结果将根据研究中的动物种类、目标脑区、注射后的经过时间、注射过程中的急性损伤量以及 i/o 接口程序的成功等因素而变化。单单位扣球活动可能不会出现, 直到1天 (在150µm 内径针的情况下) 到1周后, 注射和穗振幅可以变化多达第4-6 周。图 9显示了从一个32通道的网状电子探针中注入的具有代表性的电生理数据, 它被注射到成年雄性 C57BL/6J 鼠的海马和原体感皮层。在所有32个通道上记录了大约 300-µV 振幅局部场电位 (LFPs), 单单位扣球活动记录在26个通道上。LFPs 和孤立的峰值保持在2和4月之间的相似, 表明在这个延长的时间段内记录电子和神经元之间的高度稳定的界面。图 10显示了在注射后1年内含有网状电子的脑组织切片和染色的代表性结果。NeuN 的染色是神经胞体的标志物, 而丝是神经轴突的标记, 它在注射部位的组织密度几乎没有损失, 这意味着网状电子与脑组织之间的无缝接口。染色为 GFAP (星形胶质细胞的标记) 进一步显示近背景水平的星形胶质细胞围绕网状电子学, 表明它的存在引起很少的慢性免疫反应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58003/58003fig1.jpg"> 图 1: 注射器注射用网状电子实验中的步骤.本协议描述了使用网状电子学的典型啮齿动物神经记录实验的所有关键步骤。实验通常需要在实施的过程中 (1) 制造网状电子学, (2) 将网格电子装入毛细管针中, (3) 将网状电子的立体定向注入大脑, (4) 对网格的电 i/o 接口电子, (5) 克制或自由移动的录音, 和 (6) 网状/组织切片和染色的组织学。在一些研究中, 只有组织学数据可能需要, 在这种情况下, 步骤 (4) 和 (5) 可以跳过。<a href="https://www.jove.com/files/ftp_upload/58003/58003fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58003/58003fig2.jpg"> 图 2: 描述 ultraflexible 设备区域 (顶行)、阀杆互连区域 (中间行) 和 i/o 区域 (下一行) 的即插即用网格电子学制作程序的示意图.(A) SU-8 负光刻胶 (红色) 与 PL mask-1 图案, 以定义每个即插即用网格电子探头的底部钝化层。(B) 采用 PL mask-2、热蒸发和金属升降的模式定义了 Au 互连和 i/o 垫 (金)。(C) 采用 PL mask-3、电子束蒸发和金属升降的模式定义 Pt 电极 (蓝色)。(D) SU-8 负光刻胶 (红色) 与 PL mask-4 图案, 以确定顶部钝化层。SU-8 的开口留在每个 Pt 电极和 i/o 垫上。(E) 已完成的网格电子探针, 其中有虚线框, 指示在顶部、中间和底部行中扩大的位置。光掩模设计文件可根据作者或资源网站 meshelectronics.org 的请求提供。<a href="https://www.jove.com/files/ftp_upload/58003/58003fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58003/58003fig3.jpg"> 图 3: 即插即用网格电子的照片和光学显微镜图像.(a) 用即插即用的 i/o 对注射器注射的网状电子探针进行平铺光学显微镜图像。探测器在图 2的制造步骤完成后被成像, 但在从镀镍涂层基体上释放之前。虚线框分别从左到右对应于 C、D 和 E 中放大的 ultraflexible 设备区域、阀杆和 i/o 区域的各个部分。刻度条 = 1 毫米 (B) 3 英寸硅晶片的照片, 其中包含20个已完成的网格电子探头。刻度条 = 20 毫米 (C) ultraflexible 器件区域中20µm 直径 Pt 记录电极的光学显微镜图像。刻度条 = 100 µm. (D) 在茎区高密度 Au 互连线的光学显微镜图像。每个 Au 互连都是电隔离的, 并将单个 Pt 电极连接到一个 i/o 垫上。刻度条 = 100 µm (E) i/o 垫的光学显微镜图像。每个垫包括一个可折叠的网状区域和一个连续的薄膜区域位于茎。不导电的 SU-8 丝带连接在垫的网格部分一起帮助保持对齐。刻度条 = 200 µm.<a href="https://www.jove.com/files/ftp_upload/58003/58003fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58003/58003fig4.jpg"> 图 4: 在注射过程中举行毛细管针的装置组装.(A) 设备部件的照片。组件包括 (1) 玻璃毛细管针, (2) 吸管持有者, (3) 为吸管持有人的圆螺丝紧固件, (4) 为吸管持有者的圆锥垫圈。项目 (2) 到 (4) 包括在购买吸管持有人。箭头标记的吸管持有人的出口, 需要用环氧树脂粘合封闭。(B) 在组装和插入玻璃毛细管针后, 吸管持有人的照片。添加的环氧树脂在吸管持有者的顶端出口可见 (用箭头标记), 毛细管管将吸管持有者连接到注射器 (未显示)。(C) 将吸管持有者和毛细针的照片附在与直角端钳连接的立体定向机架后。刻度条为1厘米.<a href="https://www.jove.com/files/ftp_upload/58003/58003fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58003/58003fig5.jpg"> 图 5: 将网格电子装入玻璃针.(A) 即插即用网格电子的加载程序示意图。玻璃针位于网格电子探针的 i/o 端附近, 而悬浮在溶液中。注射器柱塞然后手动缩回, 以绘制在网格电子探针。理想的定位是与 ultraflexible 设备区域只是在针的末端。(B) 与 (a) 的照片相对应的网格电子探针被装入玻璃针。刻度条 = 2 毫米.<a href="https://www.jove.com/files/ftp_upload/58003/58003fig5large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58003/58003fig6.jpg"> 图 6: 立体定向手术站示意图.一个机动立体定向的框架 (A) 与附加的吸管持有者被用来定位针进入理想的大脑区域。针和负载网格电子的位置用物镜和附属相机 (B) 进行监测, 并显示在计算机 (C) 上。注射器泵 (D) 通过针流动精确的生理盐水, 允许精确, 控制注射网状电子进入期望的大脑区域。<a href="https://www.jove.com/files/ftp_upload/58003/58003fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58003/58003fig7.jpg"> 图 7: 即插即用 i/o 接口过程.(A) 在开颅手术的旁边, 用牙科水泥固定了一个。(B) 使用 FoV 方法将即插即用的网状电子 stereotaxically 注入所需的脑区。(C) 针, 与网格电子探针的 i/o 垫仍然在里面, 被重新定位在被控制的紧固基体之上。(D) 通过针头恢复流量, 以将 i/o 垫弹出到压紧的固定衬底上。(E) i/o 垫相对于阀杆弯曲 90°, 与导电侧朝上展开, 并在原地干燥。(F) 在 i/o 垫的边缘, 用剪刀将所述的衬底修剪成一条直线 ca. 0.5 毫米。多余的衬底被切断, 允许插入32通道的 ZIF 连接器。(G) i/o 垫片插入到安装在自定义 PCB 上的32通道 ZIF 连接器中。ZIF 连接器已锁定, 无法与 i/o 垫进行接触。(H) 闩锁被胶结关闭, pcb 被翻转到头骨上, pcb 固定在与牙科水泥的地方。(I) PCB 形成一个紧凑的 headstage 与标准的功放连接器, 便于接口在录音会话。刻度条 = 1 厘米.<a href="https://www.jove.com/files/ftp_upload/58003/58003fig7large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58003/58003fig8.jpg"> 图 8: 克制和自由移动的录音.(a) 录制会议期间, 在抑制剂中一只雄性 C57BL/6J 老鼠的照片。一个32通道的前置放大器 PCB 已插入到标准放大器接口。(B) 在自由移动录音实验中, 用32通道前置放大器印制板的同一鼠标的照片。刻度条 = 1 厘米.<a href="https://www.jove.com/files/ftp_upload/58003/58003fig8large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58003/58003fig9.jpg"> 图 9: 具有代表性的神经记录结果.(A) 有代表性的 LFP 热图, 从32通道网状电子探针注入小鼠海马体和体感皮层。数据记录, 而鼠标自由地探索其笼在2月 (顶部) 和4月 (底部) 后注射。LFP 振幅根据右边的颜色条进行颜色编码。高通滤波跟踪 (黑色) 显示的峰值活动覆盖在频谱图为每一个32通道。(B) 在 (A) 中绘制的数据排序后分离出的峰值。在32个通道的2月注射后 (顶部) 和4月注射后 (底部) 检测到26的单股扣球活动。每个峰值簇之上的数字对应于 (A) 中的通道数。这个数字已经从傅,等等。28.<a href="https://www.jove.com/files/ftp_upload/58003/58003fig9large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/58003/58003fig10.jpg"> 图 10: 代表性组织学结果.(A) 示意图, 说明网格电子在水平 (中间板) 和矢状 (下板) 脑切片中的定位。(B) 10 µm 厚皮质脑切片的荧光显微镜图像, 注射16通道电子探针后一年。切片已 immunostained NeuN (绿色)。(C) 同一脑切片 immunostained 为丝 (红色)。(D) 相同的脑切片 immunostained 为 GFAP (青色)。(E) 通过 (D) 显示网状电子/组织界面与标记的 NeuN (绿色)、丝 (红色)、GFAP (青色) 和网格电子 (蓝色) 的复合图像 (B)。刻度条 = 100 µm。这一数字已被修改从富等。27.<a href="https://www.jove.com/files/ftp_upload/58003/58003fig10large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
辅助视频 1: 对网格电子的反复加载和注入解决方案。<a href="https://www.jove.com/files/ftp_upload/58003/video1.mp4">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology at JoVE.com

Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology

网状电子探针无缝集成, 并提供稳定, 长期, 单神经元水平记录在大脑内。本协议采用网状电子技术进行体内实验, 涉及网格电子学的制作、针的装入、立体定向注射、输入/输出接口、记录实验和含有网状组织的组织学。探针。

注射器-注射用网状电子技术稳定的慢性啮齿动物电生理学

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal ...

syringe-injectable-mesh-electronics-for-stable-chronic-rodent

Research

JoVE Journal

Bioengineering

23.0K 次观看.  Harvard University. 网状电子探针无缝集成, 并提供稳定, 长期, 单神经元水平记录在大脑内。本协议采用网状电子技术进行体内实验, 涉及网格电子学的制作、针的装入、立体定向注射、输入/输出接口、记录实验和含有网状组织的组织学。探针。

视频: Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

科研

神经科学

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

清醒、无拘无束的小鼠静脉输液同时记录肾交感神经活动和血压的新方法。

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

医学

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

多柔性聚合物电极阵列的慢性植入

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

慢性活体电成像的植入系统

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...

Microinjectrode System for Combined Drug Infusion and Electrophysiology

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes and nanosensors, optimized for repeated use in awake, behaving animals. The microinjectrode system can be configured for multiple purposes: (1) simple arrangement of the cannula for placement of an experimental probe that would otherwise be too fragile to penetrate the dura mater, (2) microfluidic infusion of a drug, either independently or coupled to a cannula containing an experimental probe (i.e., microelectrode, nanosensor). In this protocol we explain the step by step construction of the microinjectrode, its coupling to microfluidic components, and the protocol for use of the system in vivo. The microfluidic components of this system allow for delivery of volumes on the nanoliter scale, with minimal penetration damage. Drug infusion can be performed independently or simultaneously with experimental probes such as microelectrodes or nanosensors in an awake, behaving animal. Applications of this system range from measuring the effects of a drug on cortical electrical activity and behavior, to understanding the function of a specific region of cortex in the context of behavioral performance based on probe or nanosensor measurements. To demonstrate some of the capabilities of this system, we present an example of muscimol infusion for reversible inactivation of the frontal eye field (FEF) in rhesus macaque during a working memory task.

We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

联合药物输液和电生理学的显微注射系统

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes ...

<meta charset="utf8"></head><body> 为临床前啮齿动物模型制造肌外肌电图电极

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode. 
Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation - ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy. 
An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 &plusmn; 0.005 mV) than uphill (0.031 &plusmn; 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 &plusmn; 0.007 mV, 0.012 &plusmn; 0.007 mV respectively; p &gt; 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

<meta charset="utf8"></head><body>作者聚焦:ACL 损伤研究中的肌外膜电极制造和测试

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

低成本肌外肌电图电极的开发:简化的制造和测试工作流程

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to ...

<meta charset="utf8"></head><body>用于大鼠多区域电生理监测的开源植入物

The TD Drive: A Parametric, Open-Source Implant for Multi&#45;Area Electrophysiological Recordings in Behaving and Sleeping Rats

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and consolidation of memories, are two examples that rely heavily on functional connectivity across the brain. In addition, investigating hemispheric similarities and/or differences goes hand in hand with these multi-area interactions. Electrophysiological studies trying to further elucidate these complex processes thus depend on recording brain activity at multiple locations simultaneously and often in a bilateral fashion. Presented here is a 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously. The open-source design was created employing parametric design principles, allowing prospective users to easily adapt the drive design to their needs by simply adjusting high-level parameters, such as anterior-posterior and mediolateral coordinates of the recording electrode locations. The implant design was validated in n = 20 Lister Hooded rats that performed different tasks. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze using two different commercial recording systems and headstages. Thus, presented here is the adaptable design and assembly of a new electrophysiological implant, facilitating fast preparation and implantation.

<meta charset="utf8"></head><body>作者聚焦：通过神经植入物破译记忆和学习以进行多区域大脑研究

Here, we present a unique, 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously.

TD Drive：一种参数化开源植入物，用于行为和睡眠大鼠的多区域电生理记录

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and ...

注射器-注射用网状电子技术稳定的慢性啮齿动物电生理学

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同时电生理记录和抑制剂在啮齿动物大脑微注射

清醒、无拘无束的小鼠静脉输液同时记录肾交感神经活动和血压的新方法。

多柔性聚合物电极阵列的慢性植入

慢性活体电成像的植入系统

联合药物输液和电生理学的显微注射系统

低成本肌外肌电图电极的开发:简化的制造和测试工作流程

低成本肌外肌电图电极的开发:简化的制造和测试工作流程

低成本肌外肌电图电极的开发:简化的制造和测试工作流程

TD Drive：一种参数化开源植入物，用于行为和睡眠大鼠的多区域电生理记录

TD Drive：一种参数化开源植入物，用于行为和睡眠大鼠的多区域电生理记录