这项工作得到了德国工业研究协会联合会 (AiF; 赠款编号: KF2780403JL3) 的赠款研究的支持。

所有协议和实验都符合目前的欧洲准则 (2010/63/欧盟), 并得到了区域当局 (Regierungspr 和 #228; sidium 和 #223; en, 20/35 先生 25/2015). 
 1. 动物 <ol> <li> 将雄性成年大鼠 (200-250 克) 置于标准实验室条件下的3-4 组, 在手术前至少一周, 以允许习服. </li>
	<li> 手术后两天后, 家鼠成对。盖上高丙烯酸盖的单笼。避免传统的金属网盖, 因为植入物可能会卡住, 增加风险, 他们成为损坏和/或不稳定随着时间的推移. </li>
</ol> 2。立体定向手术 <ol> <li> 开始手术前, 组织并准备以下设备和材料: <ol> <li> 获取无菌剪刀、钝端钳、铲子、外科手术器械快船队, 牙科钻头, 棉花芽. 和 #160; </li> <li> 获取包括异氟醚、因、盐酸曲马多、dexpantenol 眼软膏、3% 过氧化氢、聚维酮碘和70% 乙醇的药物和化学物质. </li>
		<li> 获得固定材料, 包括不锈钢螺丝、丙烯酸树脂、紫外线胶水和帽保护器. </li>
		<li> 获取微电极单元, 包括 (i) 记录单电极 (石英玻璃绝缘铂钨微电极, 锥形尖端形状, 外径:80 和 #181; m, 圆锥尖, 阻抗在1赫: 500 kOhm) 或四 (石英玻璃绝缘铂/钨4芯微电极, 外径: 100 和 #181; m, 圆锥尖端, 阻抗在1赫: 500-800 kOhm);(二) 刺激电极 (铂/铱丝 (90% 铂, 10% 铱), 芯直径125和 #181; m, 外径150和 #181; m, 阻抗和 #60; 10 kOhm) 连接到一个接触板和 (iii) 白金线参考电极 (轴直径, 100#181; 图 1A ). </li>
		<li> 获得电极支架, 用水溶性胶水粘附到微电极单元, 并预先测试至少2小时的功能 ( 图 1B ). </li>
		<li> 获取由差分前置放大器、主放大器和带通滤波器放大器组成的常规连线系统, 用于录音. </li>
		<li> 获取其他材料, 如手套、加热垫、注射器和生理盐水. </li>
		<li> 获得家庭笼子 (L x W x H:42 cm x 26 cm x 38 cm). </li>
	</ol> </li> <li> 过程 注: 在异氟醚麻醉下的常规立体定向手术中进行电极植入。
	<ol> <li> 确保实验者戴手套、手术面罩和实验室大衣. </li>
		<li> 启动麻醉将动物置于感应室 (异氟醚 4-5%, 氧气流量1升/分, 持续时间〜5分钟). </li>
		<li> 用镊子检查反射 (尾部和脚趾反射) 的损失, 以确认深层麻醉. </li>
		<li> 将动物的头部放在固定在立体定向框架上切牙棒周围的麻醉面罩中, 并调整麻醉 (异氟醚 2-3%, 氧气流量 0.7-0.8 升/分). </li>
		<li> 固定和水平排列的动物和 #39 的头部在立体定向设备使用耳棒和上切牙酒吧. </li>
		<li> 用外科快船或剪刀和聚维酮碘消毒的外科手术场. </li>
		<li> 将动物放在加热垫上以防止体温过低, 用 dexpantenol 眼膏治疗眼睛, 防止它们干燥. </li>
		<li> 注射因 (0.3-0.4 毫升, 皮下注射, 南卡罗来纳州) 在外科领域的中心. </li>
		<li> 再次测试是否有反射丢失. </li>
		<li> 用手术刀在手术场中央做一个小切口 (1.5 厘米) 来露出头骨。轻轻地将皮肤分开, 用钳子、剪刀和刮刀将残留的组织去除. </li>
		<li> 用过氧化氢包覆的棉花芽仔细清洁颅骨. </li>
		<li> 在颅骨上钻4-5 小孔 (4.7 毫米), 用于固定不锈钢螺钉. </li>
		<li> 将微电极单元/电极支架连接到前置放大器, 并附加到立体定向微 ( 图 1B 和 1C )。
		<li> 在目标区域上方的头骨上钻一个洞 (大约7毫米), 根据使用的动物的坐标从脑图集。在本研究中, 定位电极提示瞄准丘使用以下坐标, 以 bregma 为参考: 前/后, 和 #8722; 8.8 毫米;内侧/侧面, 1.5 毫米;和背/腹, 3.5 毫米 14 . </li>
		<li> 用棉芽吸收任何血液. </li>
		<li> 垂直引入微电极单元直到电极尖端到达目标区域. </li>
		<li> 将地线缆沿不锈钢螺钉和皮肤下定位. </li>
		<li> 监视尖峰活动并仔细调整电极位置与微直到到达目标结构中的活动神经元区, 并检测到适合于穗分选的信噪比的神经活动. </li>
		<li> 用紫外线胶水将微电极装置固定在头骨上, 用丙烯酸树脂覆盖接触板和螺钉. </li>
		<li> 注射生理盐水 (1 毫升的 ip) 和曲马多 (25 毫克/千克, 南卡罗来纳州), 以防止脱水, 并确保术后镇痛, 分别. </li>
		<li> 用浸在水中的刷子从电极架上断开微电极单元. </li>
		<li> 停止麻醉, 小心地将老鼠从立体定向框中移除。从微电极单元中拔下前置放大器. </li>
		<li> 在微电极单元上连接 cap 保护, 并仅在实验过程中断开. </li>
		<li> 在手术后第二天将动物在 home-cage 中保持成对. </li>
		<li> 每天监测动物的伤口感染, 体重, 健康状况和一般行为, 在手术后的7天内。在此恢复期之后, 执行 体内 电生理学和行为实验. 
		注: 手术过程持续60-90 分钟。在手术过程中, 必须连续监测和麻醉的尾巴轻弹反应, 如果需要. </li>
	</ol> </li> </ol> 3。 在体内 电生理学 <ol> <li> 设备和过程 注: 使用 TWS 进行电生理记录和刺激。
	<ol> <li> 获得具有集成前置放大器和连接电池的头阶段 (四记录通道; 模拟记录输入范围: 0-12 mV pk pk; 刺激输出: 和 #177; 625 和 #181;L x W x H:24 毫米 x 22 毫米 x 12 毫米;重量: 6 克无电池, 12 克带电池;电池运行时间可达1小时)。该头部阶段适用于通过微型多极连接器 ( 图 2 ) 直接连接到植入的微电极单元. </li>
		<li> 获取电池 (锂离子累加器, 3.7-4.2 V DC, 230 毫安, 27 mm x 20 mm x 6 mm, 1 h 操作时间) 安装在头舞台顶部 ( 图 2C )。如有必要, 可使用450毫安的更换充电电池, 约2.5 小时操作时间。在电池连接到它时, 确保在头舞台上出现绿灯. </li>
		<li> 获取通过标准 USB 端口连接到个人计算机的收发器 (接收器-发射机), 并允许无线操作多达 5 m ( 图 2E ). </li>
		<li> 获取用于电刺激和记录神经活动的 TWS 软件的个人计算机 ( 图 3 和 图 4/强>).</li>
		<li> 获取栓前置放大器和在手术过程中使用的数据采集系统 (见项目 2.1.5), 以及刺激的刺激发生器, 以便在手术后一周内对清醒大鼠 TWS 的有效进行比较. 
		注: 电刺激提供和单神经元细胞外活动记录从相同的植入微电极单位使用这两个系统。刺激参数 (电流强度, 脉冲和频率) 应根据脑区的目标调整到每个动物。在本研究中, 150-250 和 #181; 2500 赫兹电流被用来刺激下丘. </li>
	</ol> </li> <li> 行为分析 注: 一旦在收发器和动物头阶段之间没有引入金属屏障, TWS 就适用于一系列的行为任务。作为模范行为测试, 它在公开领域用于测量一般行为活动和在高加迷宫, 一个标准测试评估焦虑象行为在啮齿目动物 15 。一个摄像头被放置在开放的领域和高架迷宫的行为录音的中央。
	<ol> <li> 在进行行为测试之前, 连续三天 (每天5分钟) 处理每种动物。在每个处理周期之前, 将头阶段与电池连接到先前植入的微电极单元。在处理过程中不要进行任何记录或刺激. </li>
		<li> 打开字段 <ol> <li> 将鼠放到开放字段的中心 (40 厘米 x 40 cm x 40 厘米; 红灯 ~ 30 勒), 并允许它在神经记录下探索至少5分钟的仪器. </li>
			<li> 确定逃逸阈值-最小电流强度产生运行或跳跃。在本研究中, 提供一个高频2500赫兹刺激 (脉冲宽度: 100 和 #181; s; 脉冲间隔: 100 和 #181; s) 对下位丘在 1 min 间隔增加电流强度由20-50 和 #181; 一步, 直到老鼠表现出逃跑行为. </li>
			<li> 将鼠返回到其主笼中, 清洁野外 (0.1% 醋酸溶液) 并烘干. 
			注: 为了比较 TWS 与传统系栓系统的刺激效果, 上述过程是使用两种系统进行的. </li>
		</ol> </li> <li> 提升的迷宫 注释: 在这些实验中使用的加迷宫是由灰色丙烯酸制成, 由两个张开的手臂 (50 厘米长的 x 10 厘米宽) 和两个闭合的武器 (50 厘米长的 x 10 厘米宽, 有40厘米高的墙), 从中央 pla 延伸tform 升高50厘米, 高于地面 16 。
		<ol> <li> 将大鼠置于加迷宫的中心, 朝向一个张开的手臂, 允许它在5分钟内自由地探索持续记录下的仪器. </li>
			<li> 记录输入的条目数, 以及在5分钟内打开和关闭的武器所花费的时间. </li>
			<li> 将鼠返回到其主笼中, 清洁 (0.1% 乙酸溶液) 并在每次测试前将迷宫干燥. </li>
		</ol> </li> <li> 灌注和组织学 <ol> <li> 麻醉嗪/氯胺酮 (分别为150毫克/千克和100毫克/千克) 的鼠。
			<li> 将植入的电极单元连接到刺激电缆, 并应用电刺激 (电流强度50和 #181; A, 脉冲宽度: 100 和 #181; s; 脉冲间隔: 100 和 #181; s) 在九十年代期间, 为了在电极尖端周围产生一个小病灶. </li>
			<li> 断开刺激电缆并将动物从左心室灌注, 然后用生理盐水将200毫升的4% 甲醛在0.1 米磷酸钠缓冲液中, pH 7.3 (详细说明请参阅参考 17).</li>
			<li> 取出大脑, 在4和 #176 的新鲜定影液中浸泡4小时; C. </li>
			<li> 确保主低温室的温度在-20 和 #176; C. </li>
			<li> 在干冰上冻结大脑并将其切成50和 #181; m 系列冠状截面使用低温. </li>
			<li> 根据 Paxinos 和沃森 14 的地图集, 用 cresylviolet 着色剖面以定位电极尖端的位置. </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Rao, R. P., Mielke, F., Bobrov, E., Brecht, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vocalization-whisking+coordination+and+multisensory+integration+of+social+signals+in+rat+auditory+cortex.">Vocalization-whisking coordination and multisensory integration of social signals in rat auditory cortex.</a> Elife. 3, e03185 (2014).</li><li>Tseng, W. T., Yen, C. T., Tsai, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bundled+microwire+array+for+long-term+chronic+single-unit+recording+in+deep+brain+regions+of+behaving+rats.">A bundled microwire array for long-term chronic single-unit recording in deep brain regions of behaving rats.</a> J. Neurosci. Methods. 201 (2), 368-376 (2011).</li><li>Ball, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+scope:+a+user-configurable+digital+wireless+telemetry+system+for+freely+behaving+animals.">Rodent scope: a user-configurable digital wireless telemetry system for freely behaving animals.</a> PLoS One. 9 (2), e89949 (2014).</li><li>Chien, C. N., Jaw, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniature+telemetry+system+for+the+recording+of+action+and+field+potentials.">Miniature telemetry system for the recording of action and field potentials.</a> J. Neurosci. Methods. 147 (1), 68-73 (2005).</li><li>Hawley, E. S., Hargreaves, E. L., Kubie, J. L., Rivard, B., Muller, R. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telemetry+system+for+reliable+recording+of+action+potentials+from+freely+moving+rats.">Telemetry system for reliable recording of action potentials from freely moving rats.</a> Hippocampus. 12 (4), 505-513 (2002).</li><li>Alam, M., Chen, X., Fernandez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+multichannel+wireless+neural+stimulation+system+for+freely+roaming+animals.">A low-cost multichannel wireless neural stimulation system for freely roaming animals.</a> J. Neural. Eng. 10 (6), 066010 (2013).</li><li>Xu, S., Talwar, S. K., Hawley, E. S., Li, L., Chapin, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-channel+telemetry+system+for+brain+microstimulation+in+freely+roaming+animals.">A multi-channel telemetry system for brain microstimulation in freely roaming animals.</a> J. Neurosci Methods. 133 (1-2), 57-63 (2004).</li><li>Angotzi, G. N., Boi, F., Zordan, S., Bonfanti, A., Vato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+closed-loop+recording+and+stimulating+wireless+system+for+behaving+small+laboratory+animals.">A programmable closed-loop recording and stimulating wireless system for behaving small laboratory animals.</a> Sci. Rep. 4, 5963 (2014).</li><li>Ativanichayaphong, T., He, J. W., Hagains, C. E., Peng, Y. B., Chiao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+wireless+neural+stimulating+and+recording+system+for+study+of+pain+processing.">A combined wireless neural stimulating and recording system for study of pain processing.</a> J. Neurosci. Methods. 170 (1), 25-34 (2007).</li><li>Ye, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portable+telemetry+system+for+brain+stimulation+and+neuronal+activity+recording+in+freely+behaving+small+animals.">A portable telemetry system for brain stimulation and neuronal activity recording in freely behaving small animals.</a> J. Neurosci. Methods. 174 (2), 186-193 (2008).</li><li>Brand&#227;o, M. L., Tomaz, C., Le&#227;o-Borges, P. C., Coimbra, N. C., Bagri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense+reaction+induced+by+microinjections+of+bicuculline+into+the+inferior+colliculus.">Defense reaction induced by microinjections of bicuculline into the inferior colliculus.</a> Physiol Behav. 44, 361-365 (1988).</li><li>Brand&#227;o, M. L., Melo, L. L., Cardoso, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+defense+in+the+inferior+colliculus.">Mechanisms of defense in the inferior colliculus.</a> Behav Brain Res. 58, 49-55 (1993).</li><li>Melo, L. L., Cardoso, S. H., Brand&#227;o, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiaversive+action+of+benzodiazepines+on+escape+behavior+induced+by+electrical+stimulation+of+the+inferior+colliculus.">Antiaversive action of benzodiazepines on escape behavior induced by electrical stimulation of the inferior colliculus.</a> Physiol Behav. 51, 557-562 (1992).</li><li>Paxinos, G., Watson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotaxic coordinates. , (2007).</li><li>Walf, A. A., Frye, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+elevated+plus+maze+as+an+assay+of+anxiety-related+behavior+in+rodents.">The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.</a> Nat. Protoc. 2 (2), 322-328 (2007).</li><li>Pellow, S., Chopin, P., File, S. E., Briley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+open:closed+arm+entries+in+an+elevated+plus-maze+as+a+measure+of+anxiety+in+the+rat.">Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat.</a> J Neurosci Methods. 14, 149-167 (1985).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J. Vis. Exp. (65), e3564 (2012).</li><li>Merrill, D. R., Bikson, M., Jefferys, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+of+excitable+tissue:+design+of+efficacious+and+safe+protocols.">Electrical stimulation of excitable tissue: design of efficacious and safe protocols.</a> J Neurosci Methods. 141, 171-198 (2005).</li><li>Ewing, S. G., Porr, B., Riddell, J., Winter, C., Grace, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SaBer+DBS:+A+fully+programmable,+rechargeable,+bilateral,+charge-balanced+preclinical+microstimulator+for+long-term+neural+stimulation.">SaBer DBS: A fully programmable, rechargeable, bilateral, charge-balanced preclinical microstimulator for long-term neural stimulation.</a> J Neurosci Methods. 213, 228-235 (2013).</li></ol>

共同作者托马斯和沙克托马斯是 "托马斯录音 GmbH" 的所有者, 是开发产品使用在本研究和与本文描述的一般研究题目有关。另外, 共同作者德克 Hoehl 从 "托马斯录音 GmbH 接受收入。如果公司成功地销售与本研究相关的产品, 他们可能会从财务上获益。这项研究中使用的 "托马斯录音" 设备专门用于行为神经科学, 实验和生物心理学-Philipps 大学没有任何电荷的马尔堡。

在这里, 一个广泛的可访问的无线记录和刺激系统的电生理和行为研究在自由运动的动物提出。TWS 在行为分析中已被验证, 使用下位丘作为模型结构。TWS 方法有几个优势比现有的。首先, 该系统使用一个便携式 TWS 头阶段配备了前置放大器和充电电池, 允许稳定的长期录音多达1小时, 与相同的电池和无线操作距离长达5米. 第二, TWS 头阶段是轻和紧凑, 重量12克包括电池, 并开发, 以防止大鼠删除头部阶段和咀嚼电线。这是很好的耐受动物, 因为没有影响的 animal´s 行为剧目与和没有 TWS 头阶段被观察到, 使系统适用于一系列的行为任务。第三, 系统在 real-time 中传输。4, 通过同时进行双向记录和刺激神经活动, 该系统提供了一个复杂的工具来评估特定的大脑激活模式和行为之间的因果关系, 从而克服缺点单向系统。这一特点使该方法对深部脑刺激有特别的价值, 通常需要对长期行为实验中的刺激参数进行精确的评估、监测和调整。最后, 研制了一种可在常规立体定向手术中方便植入的集成记录、刺激和参考电极的慢性植入微电极装置。从这个角度来看, TWS 是一个综合的无线系统, 增加了刺激和记录实验的再现性。TWS 的记录质量与商业上可用的有线录音系统所产生的记录质量类似 (参见图 5)。
众所周知, 在大鼠中丘的电刺激会产生明显的逃逸行为, 其特征是奔跑或跳跃, 模仿环境挑战引起的恐惧反应11,12, 13。在本研究中, 利用 TWS 或传统系栓系统刺激下丘, 诱发了这种行为。为了测试 TWS 的刺激效果, 使用这两个系统比较了逃逸阈值--产生跑或跳的最小电流强度。TWS 头期大鼠能够快速、跳跃、跳出野外, 即显示典型的逃逸行为, 具有更大的运动自由。重要的是, 与传统的拴系系统相比, 逃逸阈值是相似的。在一起, 一个相当具有挑战性的范例被用来测试 TWS 的韧性, 它在一个无麻烦的方式中掌握。
TWS 也适合慢性电刺激实验, 因为微电极单位植入允许慢性使用。TWS 允许非常精确地调整刺激电流参数, 以便准确地检测刺激电流的频率和数量, 从而有效地引出行为反应。此外, 同样的动物被刺激与相同的电流阈值3天后和相同的期望行为反应被诱发。这表明, 刺激电极尖端周围的组织没有受到刺激电流的损害, 通常需要增加刺激电流的振幅, 并反复激励, 以引出相同的行为反应。
此外, 由于 TWS 微刺激器在图形用户界面中实时更新刺激参数, 因此可以显著缩短实验时间。其他用于临床前研究的电刺激19需要重新编程以刺激参数更新。在这些情况下, 该设备的程序是通过将动物通过电缆连接到一个编程单元。使用 TWS 时不需要这样做。
最后, 电池是固定的顶部的 TWS 头阶段和电连接到头部阶段通过一个双针磁铁连接器, 方便更换电池。其优点是, 在实验过程中, 可以改变电池, 而无需断开 TWS 头阶段从植入电极单元, 这是更舒适的动物。在目前的研究中, 我们使用的电池的操作时间只有1小时。如果实验的时间超过1小时, 建议使用额外的充电电池。TWS 可以连接到更换充电电池的容量 (i) 230 毫安为1小时操作时间或 (ii) 450 ma 约2.5 小时操作时间。两种类型的电池都可以在15分钟内完全充电。
总之, 本研究描述的操作 TWS 设计的神经刺激和记录从自由行为的小动物。本文还介绍了一套完整的植入式微电极单元、头台、接收机和软件。无线记录和刺激的质量与栓系记录系统的性能相似, 其优点是对动物更舒适、更轻便、更安全。因此, TWS 可以用来取代拴系系统, 因为它不限制动物的机动性, 并提供了一种灵活的方法, 以控制刺激和神经记录的情况下, 其他方法将是困难或不可能的。因此, TWS 可以是一个重要的工具, 以研究如何在定义的神经回路的电活动产生的某些形式的行为, 一个基本的问题在神经科学。

神经科学中的一个基本问题是, 定义的神经元电路中的电活动是如何产生某种形式的行为的。在体内电生理学是一个强有力的技术来解决这个问题, 提供一个工具来记录或刺激大脑中的电活动, 而动物正在执行某些行为任务。但是, 当前的系统经常依赖于连线电缆录制1,2, 可能会限制移动性并防止 animal´s 行为剧目的完全表达。此外, 大多数单向系统使用, 允许记录3,4,5或刺激6,7的神经活动, 但不是在同一时间或同一目标, 使它难以解开特定的大脑激活模式和行为之间的因果关系。目前, 只有少数无线、双向系统用于体内的准备工作。但是, 它们通常是重的 (40-50 克), 由两个独立的便携式单位组成,即一个头阶段和一个连接的背包为电池电源8,9,10, 使它们更少灵活和增加电缆断线的风险, 例如在打扮行为期间。上述的无线系统都没有提供植入式微电极单元, 以获得完整的 ethologically 有效行为与实验条件的高再现性完全集成的神经活动概念。
在这里, 一个新的无线, 双向设备的在体内录音和刺激神经活动在自由行为的大鼠介绍。托马斯无线系统 (TWS) 操作通过一个可移动的顶头阶段, 可以传输多通道活动使用多达四独立录音渠道和可以是目标为电脑刺激在 real-time。此外, 一个长期植入的微电极单元兼容的 TWS 开发, 既允许神经刺激和记录。本文还介绍了一种用于记录和激励的 TWS 软件图形用户界面。本研究描述了整个设备的验证和在体内的实现。
为了验证 TWS 系统, 丘被选择为目标神经结构, 因为它的电刺激可以诱发明显的行为反应。众所周知, 下丘的电刺激诱发了大鼠的 "恐惧样" 行为反应, 如警觉性、侧身姿势、背部拱起、冰冻和逃逸 (飞行) 行为。这种反应模式模仿环境挑战引起的恐惧反应, 如感知的有害事件, 攻击或威胁生存11,12,13。人们认为, 能够引出这样一个明确而明确的行为, 将给 TWS 带来真正的挑战。

<table>
	<tbody>
		<tr>
			<td>Thomas Wireless System (TWS) version 2.0</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001165</td>
			<td>The Thomas Wireless System (TWS) version 2.0 is a portable multichannel telemetry system with laptop computer, a preinstalled Microsoft Windows operating system and TWS control software. The TWS includes: low noise 4 channel pre&#8211; and programmable main amplifier with fixed bandwidth, single channel constant&#8211;current stimulator for application of biphasic current pulses, software programmable micro stimulator, implantable connector system and a basic head stage unit for mounting to an animal. The system is delivered with a transceiver with USB port connection for laptops or desktop personal computers, the control software running under Microsoft operating system Windows. The TWS system can be used for extracellular neural stimulation and recording in freely behaving small animals (e.g. rats, guinea pigs). This system can be adapted to be used in larger animals (e.g. primates) as well.</td>
		</tr>
		<tr>
			<td>Software for Thomas Wireless System (TWS)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>inlcuded in AN001165</td>
			<td>The software for the Thomas wireless system is running under Microsoft Windows operating system and provides the graphical user interface (GUI) for the Thomas Wireless System (TWS). The TWS GUI offers complete control of the TWS functions 4 channel recording and 1 channel stimulation.</td>
		</tr>
		<tr>
			<td>Implantable tetrode for recording (4 channels) and stimulation (single channel)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001132</td>
			<td>Implantable tetrode for recording (4 channels) and stimulation (single channel) for use with Thomas Wireless Systems (TWS). 
			Recording tetrode specifications: tetrode fiber material: quartz glass insulated platinum tungsten fiber, tetrode fiber outer diameter: 100&#181;m, tip shape D, impedance 0.5-0.8MOHm; Reference electrode: tip shape: D; Impedance: 300-500kOhm; Material: quartzglass insulated platinum/tungsten; Stimulation electrode specification: fiber material: platinum/iridium, diameter: 125&#181;m, lacquer insulated, tip shape : D, impedance: &#60; 10kOhm, dimensions of the electrode can be specified by the end user</td>
		</tr>
		<tr>
			<td>Implantable microelectrode for recording (single channel) and stimulation (single channel)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001118</td>
			<td>Implantable microelectrode for recording (single channel) and stimulation (single channel) for use with Thomas Wireless Systems (TWS). 
			Recording electrode specifications: electrode fiber material: quartz glass insulated platinum tungsten fiber, electrode fiber outer diameter: 80&#181;m/250&#181;m (please specify), tip shape D, impedance 0.5-0.8MOHm; Reference electrode: tip shape: D; Impedance. 300-500kOhm; Material: quartzglass insulated platinum/tungsten; Stimulation electrode specification: fiber material: platinum/iridium, diameter: 125&#181;m, lacquer insulated, tip shape : D, impedance: &#60; 10kOhm</td>
		</tr>
		<tr>
			<td>Holder for electrode implantation</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000838</td>
			<td>Special bent metal rod for microelectrode implantation for standard electrode holders. The rod is used to hold an implantable electrode. The implantable electrode is fixed to the rod with special Thomas RECORDING water soluable glue (AN001080). (Electrode holder is not included)</td>
		</tr>
		<tr>
			<td>Replacement accumulator power supply for the Thomas Wireless System (3,7V/230mAh)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001208</td>
			<td>Replacement rechargeable battery (accumulator) for Thomas Wireless System with a capacity of 230mA for approximately 1h operation time. (size: 27mm x 20mm x 6mm, weight app. 6g)</td>
		</tr>
		<tr>
			<td>Replacement accumulator power supply for the Thomas Wireless System (3,7V/450mAh)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001209</td>
			<td>Replacement rechargeable battery (accumulator) for Thomas Wireless System with a capacity of 450mA for more than 1h operation time. (size: 48mm x 30mm x 4mm, weight app. 11g)</td>
		</tr>
		<tr>
			<td>Accumulator charger for Thomas Wireless System (TWS) rechargable accumulator</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001207</td>
			<td>Mains powered charger for the Thomas Wirless System (TWS) rechargable accumulators (AN001209 and AN001209)</td>
		</tr>
		<tr>
			<td>Water soluble glue</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001080</td>
			<td>Thomas RECORDING water soluble electrode glue is a specially selected product for use with implantable microelectrodes in neuroscientific research. Its unique properties ensure a rigid connection between electrode and mounting device although it is easily removable with warm water. The Thomas RECORDING water soluble electrode glue can be used out-of-the-box, without any time consuming preparation. Thomas RECORDING water soluble electrode glue is not harmful to humans, animals or the environment. Quantity: 1 box of 10 gramms</td>
		</tr>
		<tr>
			<td>Miniature differential preamplifier</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000329</td>
			<td>The Miniature Differential Pre-Amplifier, Model MDPA-2 is a 2-channel, differential input preamplifier that is designed for low noise recordings from excitable tissue. It is intended for extracellular recording in conjunction with the implantation of implantable microelectrodes for freely moving animal appliactions with the Thomas Wireless System (TWS). The 2-Channel Miniature Differential Preamplifier (MDPA-2) is connected to the implantable microelectrodes for providing the initial tenfold amplification stage. Ideally Thomas RECORDING quartz glass insulated platinum/tungsten electrodes are used to yield optimal recording results with high signal amplitudes and low noise levels. The MDPA-2 has additional common ground and reference electrode inputs.</td>
		</tr>
		<tr>
			<td>Connection cable</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000330</td>
			<td>Connection cable to connect the Thomas Miniature differential preamplifier (MDPA-2) to a main amplifier and an accumulator power supply.</td>
		</tr>
		<tr>
			<td>Rechargeable power supply for the miniature preamplifier</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000328</td>
			<td>Rechargeable accumulator power supply for the Miniature differential preamplifier (MDPA-2).</td>
		</tr>
		<tr>
			<td>Accumulator charger (US)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000167</td>
			<td>Accumulator charger for the power supply AN000328 (US mains power outlet conenctor)</td>
		</tr>
		<tr>
			<td>Accumulator charger (EU)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000168</td>
			<td>Accumulator charger for the power supply AN000328 (EU mains power outlet connector)</td>
		</tr>
		<tr>
			<td>Differential preamplifier/main amplifier/bandpass filter</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000677</td>
			<td>TREC AC Main Amplifier (LabAmp-03) is a single-channel, differential main amplifier for neurophysiological applications (e.g. extracellular recording with microelectrodes). This Instrument is designed to work with the miniature Differential Pre-Amplifier, Model MDPA-2. The single channel of the LabAmp-03 contains a high-gain, low-noise differential amplifier stage followed by low frequency and high-frequency filters. The amplifier has two different filter amplifiers, a single unit activity (SUA) filter &#8211;amplifier and a local field potential (LFP) filter amplifier, both are connected parallel in the signal path. Record Mode offers two levels of signal gain (x10, x100) in a first stage and 4 additional levels (x5, x10, x25 and x50) in a final amplifier stage. Each amplifier has different bandpass characteristics for single unit activity (SUA) 500Hz&#8230;20kHz and local field potentials (LFP) 0,1Hz&#8230;140Hz. An audio monitor and a window discriminator is integrated in the device. The LabAmp-03 has an integrated audio monitor with loudspeaker. This unit provides audio reproduction of electrophysiological signals. The unit combines an audio amplifier in a compact, rugged package. This is especially suited to monitoring neural firing and muscle contractions. The audio monitor input is internally connected to the SUA-Filter amplifier output. The LabAmp-03 is delivered with external power supply for a mains power operation voltage range of 100-240V AC/50-60Hz.</td>
		</tr>
		<tr>
			<td>USB Oscilloscope</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001096</td>
			<td>USB PC Oszilloskop, 2 Kanal. This 2-channel PC oscilloscope is perfect suitable for mobile use on a laptop and permanent installation in control cabinets, industrial equipment and many other applications where a small, lightweight and powerful oscilloscope is required. This oscilloscope is connected to the signal output of the main amplifier is for display of recorded extracellular activity during the implanation of the implantable microelectrodes for the Thomas Wireless System (TWS). The user can acquire the measurement data over the several data-interfaces directly on the PC with includes PC software.</td>
		</tr>
		<tr>
			<td>Stimulus generator</td>
			<td>Multichannel Systems</td>
			<td>STG3008-FA</td>
			<td>Stimulus Generator for Current (STG) and Voltage Driven Stimulation fulfill three functions: current driven stimulation, voltage driven stimulation, controlling and timing. The STG is available with 2, 4 or 8 independet output channels. Featuring integrated isolation units for each output channel, the STG is able to provide any arbitrary waveform.</td>
		</tr>
		<tr>
			<td>Cap protector for the electrode</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN001193</td>
			<td>Protective cap for implantable electrode unit for the Thomas Wireless System</td>
		</tr>
		<tr>
			<td>Surgical equipment</td>
			<td></td>
			<td></td>
			<td>Scissors, blunt-end forceps, spatulas, surgical clippers, dental drill, and cotton buds</td>
		</tr>
		<tr>
			<td>Drugs and chemicals</td>
			<td></td>
			<td></td>
			<td>Isoflurane, xylocaine, tramadol hydrochloride (Tramadol-CT, AbZ-Pharma GmbH, Ulm, Germany), dexpantenol eye salve (Bepanthen, Bayer AG, Leverkusen, Germany), 3% hydrogen peroxide, povidone-Iodine (Betaisodona, Mundipharma GmbH, Limburg, Germany) and 70% ethanol;</td>
		</tr>
		<tr>
			<td>Fixation material including</td>
			<td></td>
			<td></td>
			<td>Stainless steel screws (BN650 M1.2x5; 4.7 mm ), acrylic resin (Paladur, Heraeus Holding GmbH, Hanau, Germany), ultraviolet glue (Cyberbond U3300, Cyberbond Europe GmbH, Germany) and cap protector (Thomas Recording GmbH, Giessen, Germany);</td>
		</tr>
		<tr>
			<td>Additional material</td>
			<td></td>
			<td></td>
			<td>Gloves, heating pad, syringes, and physiological saline.</td>
		</tr>
		<tr>
			<td>Small Animal Stereotaxic Instrument (SASI)</td>
			<td>Thomas RECORDING GmbH</td>
			<td>AN000287</td>
			<td>The model should be chosen according to the animal (rat, guinea pig, monkeys, etc) used in the study</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>EverFocus</td>
			<td></td>
			<td>EverFocus, model: EQ150</td>
		</tr>
		<tr>
			<td>Open field</td>
			<td></td>
			<td></td>
			<td>Made of transparent or gray acrylic, having round shape measuring 40x40x40cm</td>
		</tr>
		<tr>
			<td>Elevated plus maze</td>
			<td></td>
			<td></td>
			<td>Made of gray acrylic and consisted of two open arms (50 cm long x 10 cm wide) and two closed arms (50 cm long x 10 cm wide, with 40 cm high walls) that extended from a central platform elevated 50 cm above the floor.</td>
		</tr>
	</tbody>
</table>

a wireless, bidirectional interface for in vivo recording and stimulation of neural activity in freely behaving rats

在体内电生理学是一种强有力的技术来研究的关系之间的大脑活动和行为的毫秒和微米尺度。然而, 目前的方法大多依赖于连线电缆录音或只使用单向系统, 允许记录或刺激神经活动, 但不是在同一时间或同一目标。本文介绍了一种用于同时多通道记录和刺激自由行为大鼠神经活动的新型无线双向装置。该系统通过一个单一的便携式头部阶段, 既传送记录的活动, 也可以针对 real-time 的大脑刺激使用 telemetry-based 多通道软件。头阶段配备了前置放大器和可充电电池, 允许稳定的长期录音或刺激高达1小时. 重要的是, 头部阶段紧凑, 重量12克 (包括电池), 从而对 animal´s 的影响最小。行为剧目, 使该方法适用于广泛的一系列行为任务。此外, 该方法的主要优点是大脑刺激对神经活动和行为的影响可以同时测量, 提供了一个工具来评估特定的大脑激活模式和行为之间的因果关系。这一特性使该方法对深部脑刺激有特别的价值, 在长期行为实验中允许对刺激参数进行精确的评估、监测和调整。利用下位丘作为模型结构, 验证了系统的适用性。

TWS 技术数据
无线系统提供4独立的记录通道和1的刺激通道。通过记录的单芯电极采集细胞外活动, 并传递到无线系统的高阻抗信号输入。记录的信号是 pre-amplified (x200) 由一个 AC 耦合, 差分输入前置放大器和带通滤波 (固定信号带宽, 500 赫兹..。5赫) 只记录多活动, 因为在本研究中, 主要的兴趣是记录单位活动, 而不是局部场电位。集成可编程增益主放大器为四记录通道 (x1、x2、x4、x8、x16、x32、x64) 提供软件可调增益。无线系统的完整信号链提供了 x200、x400、x800、x1600、x3200、x6400 和 x12800 的总体增益值。在放大和滤波后, 模拟信号通过模拟到数字转换器进行数字化, 在高频载波上进行调制, 并使用 2.4-2.5 GHz ISM 波段由无线电收发机传输。在传输路径的另一侧使用了相同的收发器类型。第二台收发器通过 USB 端口连接到个人电脑。传输路径用于双向数据传输, 将来自动物的胞外记录信号发送到计算机, 反之亦然, 从计算机向动物发出信号放大和刺激的控制参数。
利用 TWS, 可以成功地记录多脑活动, 通过刺激下丘, 在野外自由运动时, 改变动物的行为。收发器被放置在离该动物5米远的位置, 并通过 USB 端口连接到计算机 (参见图 2)。在图 5中演示了与连线和无线系统所产生的记录信号质量的比较。TWS 记录多活动与一个相似的信号质量作为一个有线录音系统。微刺激器是一个真正的无线刺激, 更新刺激参数的实时, 即刺激信号, 其参数定义与 TWS 软件传递到刺激电极连接到头部阶段在一些推动刺激按钮后毫秒。因此, 可以改变刺激参数而不把动物从笼子里拿出来。这一特性的优点是可以最大限度地减少刺激实验的时间。
TWS 软件是专门设计的, 允许通过一个图形用户界面 (图 3和图 4) 控制无线系统的所有功能 (如记录和刺激)。对于微刺激, 利用 TWS 软件的图形用户界面开发了一种刺激信号。TWS 的刺激器采用了电荷平衡恒流激励模式。该刺激模式被无线发送到集成在头部阶段无线单元的恒流刺激器。刺激电流被应用在被放置在感兴趣的目标的工作微电极之间 (例如在当前研究中的下等丘) 和一个更大的遥远的柜台电极担当 TWS 的地面或参考电极。根据刺激电极阻抗和恒定电流刺激器的电压顺应性, 可以使用最大的刺激电流范围的±625µA, 虽然在目前的实验中需要更低的电流阈值。在这里, 双相电荷平衡恒流刺激用于峰值电流高达300µA。在双相刺激的情况下, 第一个脉冲被用来引出生理效应, 第二个脉冲通常反转在刺激脉冲18期间发生的电化学过程。TWS 头阶段通过 TWS 软件的图形用户界面来提供实时的刺激模式 (参见图 4)。
TWS 软件被划分成三主要部分: a (i) 主窗口与控制为录音和刺激, a (ii) 刺激生成器窗口与所有设置选择为刺激信号参量和 a (iii) 牌局窗口为重放记录的数据文件。主窗口允许用户显示多达4记录通道的记录信号, 设置所有通道的增益并开始/停止显示信号的记录。信号数据存储在计算机硬盘上的一个文件中。文件路径在 "配置" 菜单中设置。除了记录参数, 主窗口允许启动和停止的刺激过程。在主窗口屏幕上实时显示通过刺激电极在动物大脑中传递的恒定刺激电流。在刺激参数设置窗口中 pre-adjusted 了刺激信号的参数。有可能定义单或双相刺激脉冲列车, 并设置所有常用的刺激脉冲参数, 如脉冲宽度, 脉冲幅度, 脉冲之间的时间等 (详见图 4)。在激励发生器窗口的图形显示中显示了预先选定的参数值所产生的刺激脉冲函数。
TWS 软件是根据可用性方面设计的。软件的可用性是保证无线刺激/录音实验顺利进行和安全舒适工作环境的重要因素。这也有助于提高实验的重现性。
单单元记录数据和电刺激
用 TWS 和传统的栓系记录系统, 从同一个植入电极上依次记录多细胞外丘活性。图 5显示了使用两种系统记录的有代表性的原始数据, 而动物在野外自由移动。直接比较的信号表明类似的峰值波形和噪声水平 (图 5A和5B)。在 "和 B" 中描述了尖峰形式的演示。
由于大鼠在手术后没有试图移除 TWS 头阶段, 并且在随后的几天里, 它被假定它没有明显地干扰他们的运动和没有引起不适。因此, 通过使用 TWS, 避免了连接器和电缆的去除和咀嚼等问题。事实上, TWS 头阶段的大鼠能够探索野外和迷宫 (see 电影 1) 展示正常的交叉, 饲养和梳理行为。
此外, 与 TWS 或常规系栓系统所使用的刺激参数诱发相同的行为结果, 这里逃脱行为。从100µA 开始, 刺激电流振幅逐步增加, 直至达到逃逸阈值--产生跑或跳的最小电流强度, 并引出逃逸行为。在使用这两个系统时, 4 只大鼠的个体逃逸阈值是相似的 (图 5C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56299/56299fig1.jpg"> 
图 1:TWS 微电极单元(1) 记录单电极/四、(2) 刺激电极、(3) 电极纤维连接板、(4) 柔性连接电缆、(5) 地线、(6) 连接器板、(7) TWS 系统的男性或女性连接器 (A);TWS 微电极单元, 连接到前置放大器 (8) 和支架 (9);(B) 准备好附加到立体定向帧 (C)。<a href="//ecsource.jove.com/files/ftp_upload/56299/56299fig1large.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/56299/56299fig2.jpg"> 
图 2: 无蓄电池电源的 TWS 头舞台安装模块 (A) 的顶部视图。总尺寸: 高度12.5 米, 深度24毫米 (19.3 毫米 + 4.7 毫米), 宽22.1 毫米, 重量: 5.96 克. 底部视图 (B) 显示电极单元连接器;蓄能器电源, 高度 9 mm, 深度 26 mm, 宽度 20 mm, 重量 6 g (C);本测试所使用的 TWS 组件概述: (1) animal´s 头骨上装有蓄电池的头台单元, (2) 连接到计算机 USB 端口的收发器单元 (3) TWS 软件 (D);老鼠的相片自由地移动和显示 TWS 顶头阶段连接到早先被植入的微电极单位 (E) 和 TWS 软件显示模范被记录的信号 (F)。TWS 头阶段提供实时刺激模式设置的图形用户界面的 TWS 软件。<a href="//ecsource.jove.com/files/ftp_upload/56299/56299fig2large.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/56299/56299fig3.jpg"> 
图 3: TWS 软件图形用户界面, 记录屏幕.在屏幕上描述了单极性记录电极在下位丘中植入的 TWS 的记录性能。<a href="//ecsource.jove.com/files/ftp_upload/56299/56299fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56299/56299fig4.jpg"> 
图 4: TWS 软件图形用户界面刺激屏幕 (A) 和刺激参数规范 (B)。刺激信号参数 (C), 脉冲宽度 (PxW), 脉冲幅度 (PxA), 脉冲间延迟 (知识产权), 脉冲之间的时间 (TBP), 每列车 (PPT) 和时间之间的列车 (TBT) 是可调通过 TWS 软件图形用户界面。<a href="//ecsource.jove.com/files/ftp_upload/56299/56299fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56299/56299fig5.jpg"> 
图 5:用 TWS (a) 和有线录音设置 (B) 记录 extracellularly 的多信号之间的定性比较。两个录音是从相同的 TWS 微电极单元 (阻抗 0.5MOhm) 中植入的下位丘。两个记录电极接点之间的轴向距离约为400µm。有线系统和 TWS 的记录带宽相同 (500 赫兹..。5赫), 信号被取样与40赫 (有线系统) 和32赫 (TWS)。两个系统都记录了多的活动, 具有类似的信号质量。TWS 和有线录音的发射率没有明显的区别。两个记录中的神经元的动作电位波形显示在 a 和 B 中。4大鼠使用栓系系统 (TS) 或 TWS (C) 达到逃逸阈值时, 也需要类似的刺激参数。<a href="//ecsource.jove.com/files/ftp_upload/56299/56299fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie" src="/files/ftp_upload/56299/56299mov1.jpg"> 
电影 1:在加迷宫测试期间, 示范性的老鼠表现出正常的探索性行为.TWS 允许动物进入开放和闭合的武器没有导线纠缠在测试仪器, 但它是小和轻的足够, 以便它只轻微地干扰在任务本身。<a href="http://ecsource.jove.com/files/ftp_upload/56299/Movie_1_Exploratory_behavior_JoVE2017.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

Watch this Scientific Journal Video about A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats at JoVE.com

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

介绍了一种用于自由行为的大鼠多通道神经记录和刺激的无线双向系统。该系统是轻和紧凑, 因此对 animal´s 行为剧目的影响最小。此外, 这种双向系统提供了一个复杂的工具来评估大脑激活模式和行为之间的因果关系。

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

a-wireless-bidirectional-interface-for-vivo-recording-stimulation

Research

JoVE Journal

Behavior

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

13.1K 次观看.  Philipps-Universität Marburg. 介绍了一种用于自由行为的大鼠多通道神经记录和刺激的无线双向系统。该系统是轻和紧凑, 因此对 animal´s 行为剧目的影响最小。此外, 这种双向系统提供了一个复杂的工具来评估大脑激活模式和行为之间的因果关系。

视频: A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

清醒的行为小鼠慢性神经录音的微型硬盘阵列建设

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

科研

行为学

Simultaneous Scalp Electroencephalography (EEG), Electromyography (EMG), and Whole-body Segmental Inertial Recording for Multi-modal Neural Decoding

Recent studies support the involvement of supraspinal networks in control of bipedal human walking. Part of this evidence encompasses studies, including our previous work, demonstrating that gait kinematics and limb coordination during treadmill walking can be inferred from the scalp electroencephalogram (EEG) with reasonably high decoding accuracies. These results provide impetus for development of non-invasive brain-machine-interface (BMI) systems for use in restoration and/or augmentation of gait- a primary goal of rehabilitation research. To date, studies examining EEG decoding of activity during gait have been limited to treadmill walking in a controlled environment. However, to be practically viable a BMI system must be applicable for use in everyday locomotor tasks such as over ground walking and turning. Here, we present a novel protocol for non-invasive collection of brain activity (EEG), muscle activity (electromyography (EMG)), and whole-body kinematic data (head, torso, and limb trajectories) during both treadmill and over ground walking tasks. By collecting these data in the uncontrolled environment insight can be gained regarding the feasibility of decoding unconstrained gait and surface EMG from scalp EEG.

Simultaneous Scalp Electroencephalography EEG, Electromyography EMG, and Whole-body Segmental Inertial Recording for Multi-modal Neural Decoding

Development of an effective brain-machine-interface (BMI) system for restoration and rehabilitation of bipedal locomotion requires accurate decoding of user's intent. Here we present a novel experimental protocol and data collection technique for simultaneous non-invasive acquisition of neural activity, muscle activity, and whole-body kinematics during various locomotion tasks and conditions.

同时头皮脑电图（EEG），肌电图（EMG）和全身分部惯性录制多模态神经解码

Recent  studies support the involvement of supraspinal networks in control of bipedal  human walking. Part of this evidence  encompasses studies, ...

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

穿通通路 - 齿状回突触在自由活动的小鼠长时程增强

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze1,2&#160;that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.
In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal's behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.
We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

使用地面投影迷宫自动化视觉认知任务记录神经活动

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

一个过程来观察大鼠巴甫洛夫空调酒精寻求行为的背景下引发的重建

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

修改后的孔板 - 测量行为，认知和社会互动小鼠和大鼠

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

为远程沉默的神经活动在鼠害的方法在学习独立的阶段

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing &#8220;in action and in context&#8221;. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.

This protocol presents a novel methodology for the neural decoding of intent from freely-behaving infants during unscripted social interaction with an actor. Neural activity is acquired using non-invasive high-density active scalp electroencephalography (EEG). Kinematic data is collected with inertial measurement units and supplemented with synchronized video recording.

在自由，表现人类婴儿一种新的实验和分析方法意向的多模态神经解码期间社会互动

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.