Name: measuring in vivo changes in extracellular neurotransmitters during naturally rewarding behaviors in female syrian hamsters
Uploaded: 2017-09-12T14:00:00.000+00:00
Duration: 10 min 23 s
Description: Watch this Scientific Journal Video about Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters at JoVE.com

作者希望感谢本科生丹尼尔韩为他的帮助运行 Matlab 代码和本科生亚历克斯 Boettcher 为他的协助运行行为实验。这个项目是由 NSF IOS 1256799 支持 R.L.M., 并由国家卫生研究院的药物滥用的国家研究所在获奖数字 T32DA007234。

此处所述的所有程序均由明尼苏达大学机构动物保育和使用委员会 (IACUC) 批准, 并符合《实验室动物护理和使用指南》 13.
 1. 动物和插管手术 <ol> <li> 在大约55天的时间内从一个普通动物供应商获得叙利亚仓鼠. 
	注意: 虽然动物的年龄会因各种实验范式的限制而变化, 但对于性行为来说, 获得性成熟的动物是非常重要的. </li>
	<li> 在受控温度 (22 和 #176; C) 和照明 (14 h 灯后10小时暗灯亮出13:00 小时) 环境中, 与食物和水可用 ad 随意 除了在实验测试期间. 
	注: 由于仓鼠季节性繁殖, 这14:10 光/暗循环模仿它们的自然繁殖条件. </li>
	<li> 对实验室进行1周的驯化后, 在全身麻醉下进行无菌双侧 ovariectomies 和颅内立体定向 cannulations ( 如 戊巴比妥), 并管理适当的经组织批准的术后镇痛和抗生素治疗如前所述 14 , 15 . 
	注意: 对于性行为的研究, ovariectomies 是必要的, 以消除主要的内源性激素, 使动物可以统一诱导的性接受与外源激素的实验时间。
	<ol> <li> 对于多巴胺能和谷氨酸探针植入, stereotaxically 种植指南插管 (0.7 毫米直径; 图 3 )进入感兴趣的领域 ( 例如 , 本实验室以右核伏隔 (NAc) 为目标), 如在其他地方的 15 中所描述的那样。
		注: 虽然所提供的数据来自每只动物的单个谷氨酸或多巴胺探针的单传感器记录, 但该系统允许在单个动物中同时记录多达4传感器;因此, 1-4 插管可以植入根据所需的实验设计和结果。
		<ol> <li> 使用立体定向图谱 ( 例如 , 由桑色素和 #38 的金黄仓鼠大脑 ) 确定感兴趣区域的种植座坐标, 请记住, 传感器将套管下面的1毫米扩展到脑组织。
		</ol> </li> <li> 在将导管 stereotaxically 降低到所需的背腹位置后, 将其贴到头骨上, 使用由牙科丙烯酸构建的帽, 用不锈钢骨螺钉和塑料头固定 ( 图3;有关更多详细信息, 请参见 15 . </li>
		<li> 对于碳纤维测试, 插入一个参考电极 (350 和 #956; m 直径, 7.5 毫米总长度) 到对侧半球. 
		注: 不需要与套管的具体位置或距离;参考电极可以放置在对侧半球方便的任何地方. </li>
	</ol> </li> </ol> 2。生物传感器和碳纤维检测 <ol> <li> 手术后1周的恢复, 管理激素启动方案, 诱导性接受。
	<ol> <li> 注入10和 #181; 在0.1 毫升棉籽油皮下 (南卡罗来纳州), 约48ĥ和 24 h 雌二醇 g, 在性行为测试之前, 对男性的性接受度为 11 。
		<li> 注入500和 #181; 0.1 毫升棉籽油中的黄体酮 g, 在引入雄性诱导性接受前 4 h. 11 . </li>
	</ol> </li> <li> 在其每日循环的黑暗阶段开始时测试所有动物, 因为啮齿动物的社会行为会在白光和红光测试条件下受到改变 16 17 , 18 . </li> <li> 用于酶生物传感器的谷氨酸测试, 校准传感器 体外 ( 图 4 ), 然后使用之前所述 15 , 19 . <ol> <li> 使校准解决方案在20毫升玻璃离心管的测试中更加新鲜。对于分析溶液, 将7.4 毫克 l-谷氨酸分解为10毫升的超纯 H 2 O 通过轻轻倒置管。使干扰溶液溶解176.1 毫克 AA 在10毫升的超纯 H 2 O. </li>
		<li> 通过在基本实验室环架底座上放置磁力搅拌器来设置校准。在磁力搅拌器的顶部放置一个20毫升的夹套烧杯, 并使用中型2爪钳夹住。
		<ol> <li> 将磁性搅拌棒和20毫升的100毫米磷酸盐缓冲盐水 (PBS) 添加到夹套烧杯中。将夹套烧杯连接到循环水浴, 将缓冲液加热到37和 #176; C. 
			注意: 酶生物传感器的校准必须在体温下进行, 因为酶活性受温度影响. </li>
		</ol> </li> <li> 将传感器校准托架放在夹套烧杯的顶部, 并在顶部设置4通道校准前置放大器, 使用直角钳固定到位。将前置放大器连接到数据调理和采集装置, 以记录校准数据. </li>
		<li> 测试每个酶生物传感器对谷氨酸 (或其他商业可用生物传感器探头的其他分析) 的灵敏度, 例如, 在实验录制之前, 将探针放置在夹套烧杯, 淹没传感腔和部分氯化银 (氯化) 参考线在缓冲液中。
		<ol> <li> 在将每个传感器 (最多 4) 连接到4通道校准前置放大器的端口上之前, 在计算机接口上使用免费下载的采集软件启动新的记录。允许传感器达到稳定的基线. </li>
		当传感器到达稳定基线时, </ol> </li> <li> 开始添加分析物注入。使用校准架上的孔将吸管尖端引入缓冲液。使用 "录制软件" 中的 "快速密钥注释" 工具在进行注入时进行批注。
		<ol> <li> 使10和 #181; M 将移40和 #956 的谷氨酸添加到缓冲区解决方案中。等待传感器在加法之间稳定。添加3注射的分析物溶液, 然后加入干扰 AA 溶液 (50 和 #956; 250 和 #956 的注入量; M 浓度的变化). 
			注: 校准允许转换安培信号的变化 (在步骤2.12 中的酶实验记录中看到), 以改变谷氨酸浓度, 如果需要。有关逐步指令, 请参见参考 15 。该实验室使用的多巴胺能探针在制造过程中由商业供应商进行校准。谷氨酸探针必须在使用时进行校准, 因为它们的灵敏度随着时间的推移而降低, 因为酶的成分会降低 19 。由于碳纤维电极不经过降解, 该公司在装运前进行的校准就足够了。这里是增加的可能性损坏稀薄的碳纤维 (350 和 #956; m 直径) 通过另外的处理. </li>
		</ol> </li> </ol> </li> <li> 从动物和 #39 的家笼中取出带床上用品的测试室. 
	注意: 这增加了女性和 #39; 对测试室的熟悉程度, 以及暴露雄性对雌性在雌二醇治疗中散发的性刺激嗅觉交配的提示. </li>
	<li> 在插入探针之前, 用异氟醚或在诱导室中的另一种挥发性麻醉剂轻轻麻醉动物. </li>
	<li> 从导套管中取出咬合 obdurator, 然后在导向轴上插入碳纤维电极或酶探针. </li>
	<li> 探头插入后, 将动物放在测试室中. 
	注: 本实验室使用10加仑玻璃水族箱 (24.5 x 48.9 x 29.4 厘米), 但也可采用相等面积的圆形腔室. </li>
	<li> 开始在商业上可用的软件程序中记录视频和 time-locked 安培信号, 在其他地方详细描述 15 . </li>
	<li> 将传感器连接到记录系统: 电位通过电屏蔽电缆和电动旋转。通过拧紧固定在头上的针脚, 稳定传感器连接。必要时使用实验室胶片加强连接. 
	注: 需要一个定制的连接器来连接碳纤维电极和参考电极的电位, 而酶谷氨酸传感器可以直接连接到电位. </li>
	<li> 在实验测试前允许传感器在大脑中平衡. 
	注意: 平衡时间的不同取决于记录传感器 ( 例如 , 酶传感器需要 2-4 h 平衡, 而碳纤电极只需要大约30分钟)。如果两种类型的传感器被植入, 平衡的更长的酶传感器时间. </li>
	<li> 在传感器平衡后, 将刺激男性引入测试室. </li>
	<li> 在第一次安装后, 由刺激男性插入 (称为 intromission), 继续录制额外的 10-30 分钟, 具体取决于实验目标. </li>
	<li> 在进行行为测试后, 断开女性和 #39 的传感器. </li>
	<li> 将两个仓鼠从测试室中取出。使用70% 乙醇去除床上用品和清洁房间. 
	注: 由于碳纤维记录所需的短暂平衡, 步骤 2.3-2.14 可以重复, 以测试多个动物在同一天。相比之下, 由于酶传感器需要大约 4 h 的平衡, 每天只测试一只动物, 以限制可能的学科变异由于动物和 #39 的差异; 昼夜时间在测试. </li>
</ol> 3。牺牲和灌注 <ol> <li> 在测试期结束后, 用安乐试剂 ( (如 , 本实验室使用腹腔注射0.2 毫升苯妥英和戊巴比妥溶液) 深入麻醉女性试验对象。.</li>
	<li> Transcardially 灌注的动物与25毫米 PBS, pH 7.6, 以消除所有循环血液, 其次是20分钟的固定使用4% 甲醛 pbs. </li>
	<li> 在一夜间50毫升锥形管中, 将4% 甲醛溶液中的30毫升的大脑和后缀移除。
	<ol> <li> 将大脑存储在10% 蔗糖-PBS 溶液中, 直到准备好串行部分. 
		注意: 大脑可以储存2周, 每周进行蔗糖溶液的改变, 以避免污染物的潜在生长. </li>
	</ol> </li> <li> 串行部分40和 #181 中的大脑; m 切片通过植入区域, 如前面所描述的冻结切片或低温 19 。
	<li> 在商业上可用的粘附涂层幻灯片上连续安装切片 (或参见 20 进行)。允许干燥, 至少在一夜. </li>
	<li> 用甲酚紫色染料、清除和片着色幻灯片, 如前所述 20 . </li>
	<li> 使用明亮的现场显微镜对幻灯片进行图像验证, 以确认传感器的解剖位置. </li>
</ol> 4。行为编码 <ol> <li> 使用免费的商用可下载软件查看和注释视频 (参见 材料的表 ) 慢动作, 以精确的代码行为 time-locked 安培信号. 
	注意: 要在分析中使用 MATLAB 代码 (见下面的 ) , 请对每个女性和 #39 的 开始 和 结束 分别进行批注; s 和 #39 的行为。
	<ol> <li> 在框架中对 startLordosis 进行批注时, 雌性启动了 dorsoflexion 的背部并将其尾部向上缺陷。当女性终止这种姿势时, 当雌性调整到测试室的另一个位置时, 通常会发生这种情况. </li>
		<li> 批注 startAI 当雌性处于前姿势时, 雄性会将他的鼻子移向雌性和 #39; 生殖器区域, 在那里可能会发生嗅吸、舔舐或鼻子她的会阴区域。注释 endAI 当雄性从靠近雌性和 #39 的生殖器区域移开他的鼻子时. </li>
		<li> 批注 startMount 当男性接近并将其前爪置于女性的位置时, 无论安装尝试的方向如何 (如 、侧面、臀部). </li>
		<li> 批注 startIntromission 当成功的插接时, 会使安装的男性生殖器进入女性和 #39 的阴道. 
		注: intromission 是行为可区分的, 在 intromission 之前发生, 男性明显拉他的上肢对他的身体, 推力他的骨盆向前, 并卷曲他的尾巴向上, 表明渗透 (见强类 = "xfig" > 图 4 C )。
		<ol> <li> 如果适用, 则批注 射精 。
			注意: 在交配回合结束时, 与成功的 intromission 结合, 雄性会射精。虽然你无法想象实际的排放, 雄性仓鼠有一个典型的踩踏运动与他们的后腿在 intromission 21 , 从而注释的射精发生的脚运动. </li>
		</ol> </li> <li> 批注 endIntromission 和 endMount 同时在框架中, 当男性已经删除了他的前两个爪子, 因此, 没有与女性的接触。由于经常无法想象他的撤退, 同时编码这两种行为, 以使一致性和可靠性跨交配回合. 
		注意: 交配回合要么遵循一个成功的射精, 要么是当女性打破了前的姿势后, 至少有一个 mount . </li>
	</ol> </li> </ol> 5。示例数据分析 注意: 使用固定电位碳纤和酶记录多巴胺 a谷氨酸, 分别, 允许的能力, 以测量的补品和阶段性模式的电化学释放。该实验室使用一个免费的商业采集系统和软件来记录和转换从模拟到数字的记录信号 (偏差 0.600 V, 采样率 = 1 Hz). 
<ol> <li> 示例数据分析协议的说明 <ol> <li> 尽管可以使用其他程序, 但用 MATLAB 对记录的电化学信号中的主音和相位 (瞬态) 变化进行量化。
		<ol> <li> 对于所提供的数据, 使用50点移动平均滤波器计算主音信号。通过从原始数据中减去主音信号, 计算出信号的瞬态唯一归一化表示。在识别相位峰值时, 有各种算法选项 ( 例如 , 将峰值标记为超过平均值 1 SD 的信号的变化). </li>
			<li> 确定这些峰值的位置, 即 , 局部极大值, 从正常化信号使用 MATLAB 函数 peakfinder , 将最小峰值振幅设置为信号的根平均平方。从归一化信号中检测出峰值的位置, 提取各峰值的振幅。检测峰值的能力允许识别任何可能与之相符的 time-locked 行为, 或在被调查的神经中驾驶相对峰值. </li>
		</ol> </li> <li> 按照行为注释对处理的数据进行分段 (请参见步骤 3); 学生和 #39; s t 测试用于评估强直性神经递质水平、瞬变的发生和振幅在交配回合期间, pre-mating 回合前与前的瞬变。为了测量电化学释放的补剂模式的变化, 在交配回合 ( 即 , pre-mating 回合前) 之前, 在 前 位置直接发生的数据点与交配回合中的数据点进行了比较。使用 t 测试的会话. </li>
	</ol> </li> <li> 为所描述的数据分析协议准备原始数据 注: 如上所述, 虽然其他程序或分析可能是同样可行的选项, 但为了使用上面描述的 MATLAB 代码, 必须准备原始数据以下方式: <ol> <li> 导出注释和电压文件。要做到这一点, 在 #39; 文件和 #39; 选项卡上, 选择 #39; 导出和 #39; 函数并选择 #39; 批注和 #39; 选项卡以保存批注。在 "#39; 文件和 #39; 选项卡上, 选择 #39; 导出和 #39; 函数并选择 #34; TSV 和 #39; 选项卡将电压测量保存为 TSV 扩展文件. </li>
		<li> 使用电子表格程序打开每个文件并保存为和 #39; xls/xlsx 和 #39; 使文件与 MATLAB 兼容. 
		注: 对于和 #39; 注释和 #39; 文件, 还有其他注释必须在电子表格程序中手动添加 post-export, 以口述 pre-mating 前的 开始 和 end (批注为 PreMBlordorsis ) 和交配回合, 使这些时间点可以直接比较, 在步骤5.1.2 中提到. </li>
		<li> 注释 pre-mating 回合前 ( startPreMBlordosis ) 的开始, 同时前行为开始 ( startLordosis ) 也包括 装入 行为, 并且在新的交配回合开始时, 如果女性保持在前的姿势. </li>
		<li> 在第一个 装入 开始时, 对 pre-mating 回合前 ( endPreMBlordosis ) 的末尾进行批注. </li>
		<li> 在 endPreMBlordosis 的同时对交配回合 ( startMB ) 的开始进行注解, 因为这意味着交配回合的开始. </li>
		<li> 在 装入 包括 射精 (请参见步骤 4.1.4.1) 的末尾 (endMB) 或在前姿势 ( endLordosis ) 的终止后, 对交配回合的结束进行批注. </li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

虽然相对简单, 但在使用此技术时可能会出现一些问题。首先, 探针的立体定位必须是精确的: 不像微透析, 样本更广泛的细胞外环境半径周围的探针, 这种技术只允许测量的神经递质进入直接接触用探针其次, 在碳纤维记录的情况下, 由于纤维的宽度小, 断裂可能发生, 并且探针必须插入与故意关心。在谷氨酸生物传感器的情况下, 如果探针不能在3周的保证时限内使用, 就会发生酶降解。幸运的是, 所...

在清醒行为的动物中测量神经递质释放的能力使研究人员能够将特定行为与神经的时空模式联系起来--这是研究自然的机制和电路的有力工具。和 real-time 的行为。从历史上看, 微透析用于测量脑细胞外环境中的电反应和反应物质1。这项技术使用一个类似的离子组成的水溶液的连续流动的胞外流体, 通过一个微透析探针组成一个小轴与尖端制成的透中空纤维膜2。在插入探针后, 神经递质或其他分析的利益可以通过被动扩散在透膜之前被收集, 然后用高效液相色谱法 (HPLC) 进行分析,分析化学技术通常用于分离、识别和量化异构混合物中的组分3。
虽然微透析是一种敏感的技术, 可以用来测量几乎任何感兴趣的分析物, 时间分辨率很低, 最大采样率在几分钟到几十分钟的顺序1,2。快速扫描循环伏安法 (FSCV) 的发明, 一种依赖于活性种的氧化还原电位的技术, 可以在细胞外液中的感兴趣的分析物的瞬时浓度附近进行澄清。简要 (参见罗宾逊et al.4为了进行广泛的审查, 在快速的时间刻度4中, 使用一个电极来提高和降低三角波的电压。当电压在正确的范围内, 复利的化合物被反复氧化和减少。这种氧化和还原导致电子的运动产生一个小的交流电流。扫描率发生在秒的规模与氧化和减少的化合物发生在微秒。通过从产生的电流中减去探针所产生的背景电流, 就可以产生电压与每个化合物所特有的电流图。由于电压振荡的时间尺度是已知的, 这些数据可以用来计算作为一个时间函数的电流的情节。因此, 只要在每次氧化和还原反应中转移的电子数已知为4, 就可以确定化合物的相对浓度。
这种化学特异性和高的时间分辨率使 FSCV 成为一种强有力的技术来检测变化的化学浓度在体内。然而, 尽管有这些多方面的优势, 这项技术需要广泛的技术专长和昂贵的设备和设置。此外, nonelectroactive 神经递质 (例如, 谷氨酸) 不能用这种技术来衡量。幸运的是, 在电化学领域的技术进步5, 以及这些发明的商业化, 引入了一个相对简单的方法来测量清醒行为动物的 non-electroactive 神经递质不影响时间精度--一种称为酶生物传感器技术的技术。这种技术使用酶的 nonelectroactive 神经递质转化为两个底物, 其中之一是活性过氧化氢被检测为安培氧化电流产生的应用电位5.商用生物传感器探头 (参见图 1) 通过竞争性地减少内源 interferents 的贡献, 有选择地测量分析的兴趣。在谷氨酸的情况下, 共同的干扰抗坏血酸 (aa) 的贡献是竞争性地减少到被测量的电流由 co-localizing aa 氧化酶在传感器的活跃酶表面上, 转换 aa 为 non-electroactivedihydroascorbate 和水。此外, 在酶层下存在的负电荷磺聚合物层排除了内源性阴离子化合物。
这种生物传感器实验装置可以测量活性神经递质, 如在 FSCV, 但它采用了固定电位记录6。与在 FSCV 中应用的振荡电压相反, 在固定电位记录中, 电压保持在对感兴趣的分析物的氧化还原电位上。尽管它的化学选择性比 FSCV 低, 因为多个神经递质可能具有相同的氧化还原潜能, 但在绝大多数情况下, 大脑区域的神经递质是扭曲的, 这种方法的交钥匙性质胜过了缺乏化学特异.
能够测量活性和 nonelectroactive 神经递质释放在近 real-time, 并链接到特定的行为事件提供了一个机会, 研究收敛神经递质释放。这份手稿详细介绍了这一系统的使用, 以询问多巴胺和谷氨酸神经响应自然奖励在清醒行为仓鼠。本文的目的是详细介绍在雌性仓鼠性行为期间, 测量这种神经递质释放的过程, 目的是展示其检查其他行为和实验范式的可行性。
仓鼠是用于电化学记录的理想模型 历史上, 老鼠和老鼠模型在性行为的研究中被使用。这些啮齿类动物参与一个动态的交配序列, 涉及许多女性邀约行为, 包括跳跃, 穿梭, 和耳朵摆动引诱男性追逐, 并最终安装女性7。由男性装载 (有或没有阴道渗透) 仅持续几秒钟, 在期间女性参与她的性行为姿态 (被称为前) 也仅几秒钟在恢复活跃邀约行为之前。这种行为模式, 由高水平的活动穿插, 短暂的不动的时期, 是一个问题, 以测量神经行为的动物。首先, 在与神经活动无关的安培记录中可以有运动工件。第二, 运动与特定神经递质在某些脑区的释放有关。例如, 多巴胺释放已被耦合到背部和腹侧纹状体的运动活动8,9, 这一发现构成了微透析测量多巴胺后精神的基础管理10。因为在 mos 中女性典型的邀约行为啮齿类动物涉及高水平的运动活动, 并由10分钟的性行为测试的大部分, 这使得很难把神经的变化归咎于性行为的显式成分, 集体最后只分钟.
为了分析女性性行为的神经特征, 该实验室寻找了一个在性行为中有最小运动活动的物种。交配序列在叙利亚仓鼠 (Mesocricetus 鲫) 是理想的神经录音由于缺乏邀约行为通常见于老鼠和老鼠11。因此, 雌性仓鼠将进入并保持前的姿势, 在10分钟的测试会话12中超过9分钟。由于女性缺乏外来的运动动作 ,在体内可以获得与雄性互成分相关的电化学记录。
仓鼠交配回合 在将雄性刺激动物引入试验室后, 雄性将在安装前先进行雌生殖器调查 (AI) (图 2a)。为了雄鸟的坐骑, 雌鸟必须接受一种被称为前的性体位, 在这一姿势中, 她会拱起背部, 使其尾巴偏转, 这样, 雄性就能获得阴道的生殖器。雄鸟会装上雌性, 用两只爪子 (图 2B) 将她的后躯紧握起来, 并开始尝试获取 intromission (图 2C)。雄鸟在最终达到射精前, 会将雌性 (不插入) 和 intromit 多次安装在一起。这一系列的坐骑和 intromissions 导致射精被称为 "交配回合"。男性将有几个交配回合在一个单一的会议。

<table><tbody><tr><td>Nembutal</td><td>Oak Pharmaceuticals Inc.</td><td>76478-501-50</td><td>Pentobarbital sodium injection, USP. This lab uses 8.5 mg/100 g body weight, injected intraperotineally.&amp;nbsp;</td></tr><tr><td>Loxicom analgesic&amp;nbsp;</td><td>Norbrook Laboratories&amp;nbsp;</td><td>6451603670</td><td>NSAID antinflammatory and analgesic used for post-operative pain control. Generic: meloxicam.&amp;nbsp;</td></tr><tr><td>Enroflox antibiotic&amp;nbsp;</td><td>Norbrook Laboratories&amp;nbsp;</td><td>5552915411</td><td>Fluoroquinolone antibiotic for post-operative infection prevention. Generic: Enrofloxacin.</td></tr><tr><td>Beuthanasia-D&amp;nbsp;</td><td>Merck Animal Health</td><td>00061047305</td><td>Pentobarbital Sodium, Phenytoin Sodium euthanasia agent.</td></tr><tr><td>Bone screws</td><td>Pinnacle Technologies, Inc.</td><td>8111-16</td><td>1/8&quot; bone screw (Pkg. of 16) used to affix skull cap to skull.&amp;nbsp;</td></tr><tr><td>Dental acrylic (Bosworth Duz-All)</td><td>Bosworth&amp;nbsp;</td><td>166261C&amp;nbsp;</td><td>Self curing dental acrylic is used in construction of a skull cap to affix cannula and head mount to skull.</td></tr><tr><td>Hardware biosensor setup&amp;nbsp;</td><td>Pinnacle Technologies, Inc.</td><td>8400-K2</td><td>Pinnacle offers complete hardware kits for new users of our tethered biosensor system for rats. Kits include a commutator, preamplifier, and data conditioning and acquisition system</td></tr><tr><td>Base video computer package</td><td>Pinnacle Technologies, Inc.</td><td>9000-K1</td><td>The base computer package includes a preconfigured computer with ample hard disk storage, a high-definition monitor, a keyboard and mouse, an uninterruptible power supply, and all necessary cables.&amp;nbsp;</td></tr><tr><td>Video EQ700 EverFocus camera&amp;nbsp; package</td><td>Pinnacle Technologies, Inc.</td><td>&amp;nbsp;9000-K10&amp;nbsp;</td><td>EQ700 night vision capable box camera with independent IR source was obtained as part of Pinnacle video computer package. Dome camera (9000-K9) and HD camera (9000-K11) options are also available.&amp;nbsp;</td></tr><tr><td>Sirenia Acquisition software</td><td>Pinnacle Technologies, Inc.</td><td>Free--available to download from pinnaclet.com</td><td>Sirenia&amp;nbsp;Acquisition provides a single platform for recording data from any Pinnacle hardware system. The software features synchronization of all data streams, user-configurable settings, data consolidation, and multiple export options. In addition, the software includes basic review and analysis modules for biosensor recordings. Sirenia&amp;nbsp;delivers free ll-in-one software that is ideal for data acquisition and review.</td></tr><tr><td>Tethered rat&amp;nbsp;in vitro&amp;nbsp;calibration kit</td><td>Pinnacle Technologies, Inc.</td><td>7000-K2-T-BAS&amp;nbsp;</td><td>In order to relate the current changes measured by a biosensor to actual changes in analyte concentration, it is necessary to calibrate the biosensor prior to implantation into the animal. The process also confirms the integrity and selectivity of the sensors.&amp;nbsp;Calibration kit includes 20 mL jacketed beaker (#7058), 1/2&quot; by 1/8&quot; magnetic stir bar (#7059), right angle clamp (#7056), 2 prong single-adjustment clamp (#7055), 4-channel calibration preamplifer (#7053), and calibration holder (#7051).&amp;nbsp;</td></tr><tr><td>Stir plate&amp;nbsp;</td><td>Corning</td><td>6795-410D</td><td>Corning digital Stirrer, 5&quot; x 7&quot;, 120 VAC used to spin magnetic stirrer in jacketed beaker during &lt;em&gt;in vitro&lt;/em&gt; calibration of glutamate biosensors.</td></tr><tr><td>Water bath capable of closed loop circulation</td><td>PolyScience</td><td>8006A11B</td><td>PolyScience 8006A11B 6L Standard Digital Heated Circulating Bath, 120VAC water bath was used with plastic tubing to heat jacketed beaker to physiological temperature.&amp;nbsp;</td></tr><tr><td>Carbon fiber sensor with BASi rat cannulae</td><td>Pinnacle Technology, Inc.</td><td>7002-CFS</td><td>Carbon fiber electrode used for recording dopamine neurotransmission.</td></tr><tr><td>Ag/AgCl reference electrode</td><td>Pinnacle Technology, Inc.</td><td>7065</td><td>Necessary for carbon fiber recordings.</td></tr><tr><td>Glutamate biosensors&amp;nbsp;</td><td>Pinnacle Technology, Inc.</td><td>7001&amp;nbsp;</td><td>Enzymatic biosensor probe used for recording glutamatergic neurotransmission.</td></tr><tr><td>BASi guide cannulae</td><td>Pinnacle Technologies, Inc.</td><td>7030&amp;nbsp;</td><td>Guide cannulae implanted into brain region of interest to guide probe.</td></tr><tr><td>BASi cannula plastic headpiece for rats&amp;nbsp;</td><td>Pinnacle Technologies, Inc.</td><td>7011&amp;nbsp;</td><td>Headmount stabilizes probe and attaches to potentiostat.</td></tr></tbody></table>

measuring in vivo changes in extracellular neurotransmitters during naturally rewarding behaviors in female syrian hamsters

在快速的时间尺度上测量神经递质释放的能力允许神经的模式与特定的行为或操纵相联系;阐明底层机制和电路的有力工具。虽然微透析技术已被用于几十年来测量几乎任何对大脑感兴趣的分析物, 但这种技术在时间分辨率上是有限的。另外, 快速扫描循环伏安法既精确又灵敏;然而, 由于这一技术上困难的方法依赖于 electroactivity 的分析物的兴趣, 有可能检测 nonelectroactive 物质 (例如, 神经递质谷氨酸) 被消除。本文详细介绍了结合固定电位安培和酶传感的交钥匙系统, 用于测量活性和 nonelectroactive 神经递质的时间精度。这两种强大的技术配对, 使测量的补品和相位神经相对轻松, 并允许记录多个神经递质同时。这篇手稿的目的是演示在雌性仓鼠中使用自然奖赏行为 (即, 性行为) 来测量多巴胺和谷氨酸神经在体内的过程, 最终目标是显示本试验的技术可行性, 用于检测其他行为和实验范例。

利用上述的电化学和行为编码方法, 该实验室已经开始在体内性行为的记录中, 对多巴胺和谷氨酸中的补品和阶段性波动进行表征。由于这个方法的世俗地确切的方式, 我们能更加准确地刻画神经在性行为期间;以及在交配回合期间, 将特定的释放模式变化归因于相应的补药变化, 以及在女性接受个体交配行为期间的相应的瞬时波动 (例如, intromission, 射精)从安装?...

Watch this Scientific Journal Video about Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters at JoVE.com

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

本文详细介绍了使用碳纤电极和酶生物传感器技术的固定电位安培记录, 以测量在自然奖励行为中的高时间分辨率的多巴胺和谷氨酸的释放。雌性仓鼠

雌性叙利亚仓鼠自然奖赏行为中细胞外神经递质的测量体内变化

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

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Introduction to Psychology

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Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Biopsychology

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10.1K 次观看.  University of Minnesota. 本文详细介绍了使用碳纤电极和酶生物传感器技术的固定电位安培记录, 以测量在自然奖励行为中的高时间分辨率的多巴胺和谷氨酸的释放。雌性仓鼠

视频: Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

转录动力学的脑组织样本在行为经验综合分析

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

科研

行为学

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 ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

量化的社会动机小鼠使用操作条件反射

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

在啮齿类动物的存活立体定向手术

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

生物学

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

雄性大鼠寻救行为的冲突模型

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

同时监测无线电生理学和记忆行为测试作为研究海马神经发生的工具

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

中脑多巴胺神经元的数目在成年小鼠环境的调变

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

神经科学

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

从脑边缘和Mesocortical多巴胺奖赏网站继朴素的糖和脂肪摄入大鼠c-fos激活的同时检测

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

雌性叙利亚仓鼠自然奖赏行为中细胞外神经递质的测量体内变化

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