Name: measuring in vivo changes in extracellular neurotransmitters during naturally rewarding behaviors in female syrian hamsters
Uploaded: 2017-09-12T14:00:00
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)...

<ol>
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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。

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			<td height="42" style="height:42px;width:329px;">Nembutal</td>
			<td style="width:183px;">Oak Pharmaceuticals Inc.</td>
			<td style="width:323px;">76478-501-50</td>
			<td style="width:504px;">Pentobarbital sodium injection, USP. This lab uses 8.5mg/100g body weight, injected intraperotineally.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Loxicom analgesic&#160;</td>
			<td style="width:183px;">Norbrook Laboratories&#160;</td>
			<td style="width:323px;">6451603670</td>
			<td style="width:504px;">NSAID antinflammatory and analgesic used for post-operative pain control. Generic: meloxicam.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Enroflox antibiotic&#160;</td>
			<td style="width:183px;">Norbrook Laboratories&#160;</td>
			<td style="width:323px;">5552915411</td>
			<td style="width:504px;">Fluoroquinolone antibiotic for post-operative infection prevention. Generic: Enrofloxacin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Beuthanasia-D&#160;</td>
			<td style="width:183px;">Merck Animal Health</td>
			<td style="width:323px;">00061047305</td>
			<td style="width:504px;">Pentobarbital Sodium, Phenytoin Sodium euthanasia agent.</td>
		</tr>
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			<td height="21" style="height:21px;">Bone screws</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>8111-16</td>
			<td style="width:504px;">1/8&#34; bone screw (Pkg. of 16) used to affix skull cap to skull.&#160;</td>
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			<td height="42" style="height:42px;">Dental acrylic (Bosworth Duz-All)</td>
			<td>Bosworth&#160;</td>
			<td>166261C&#160;</td>
			<td style="width:504px;">Self curing dental acrylic is used in construction of a skull cap to affix cannula and head mount to skull.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Hardware biosensor setup&#160;</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>8400-K2</td>
			<td style="width:504px;">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>
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			<td height="60" style="height:60px;">Base video computer package</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>9000-K1</td>
			<td style="width:504px;">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.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Video EQ700 EverFocus camera&#160; package</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>&#160;9000-K10&#160;</td>
			<td style="width:504px;">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.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Sirenia Acquisition software</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>Free--available to download from pinnaclet.com</td>
			<td style="width:504px;">Sirenia&#160;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&#160;delivers free ll-in-one software that is ideal for data acquisition and review.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;">Tethered rat&#160;in vitro&#160;calibration kit</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7000-K2-T-BAS&#160;</td>
			<td style="width:504px;">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.&#160;Calibration kit includes 20 mL jacketed beaker (#7058), 1/2&#34; by 1/8&#34; magnetic stir bar (#7059), right angle clamp (#7056), 2 prong single-adjustment clamp (#7055), 4-channel calibration preamplifer (#7053), and calibration holder (#7051).&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Stir plate&#160;</td>
			<td>Corning</td>
			<td>6795-410D</td>
			<td style="width:504px;">Corning digital Stirrer, 5&#34; x 7&#34;, 120 VAC used to spin magnetic stirrer in jacketed beaker during in vitro calibration of glutamate biosensors.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Water bath capable of closed loop circulation</td>
			<td>PolyScience</td>
			<td>8006A11B</td>
			<td style="width:504px;">PolyScience 8006A11B 6L Standard Digital Heated Circulating Bath, 120VAC water bath was used with plastic tubing to heat jacketed beaker to physiological temperature.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbon fiber sensor with BASi rat cannulae</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7002-CFS</td>
			<td style="width:504px;">Carbon fiber electrode used for recording dopamine neurotransmission.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ag/AgCl reference electrode</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7065</td>
			<td style="width:504px;">Necessary for carbon fiber recordings.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Glutamate biosensors&#160;</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7001&#160;</td>
			<td style="width:504px;">Enzymatic biosensor probe used for recording glutamatergic neurotransmission.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BASi guide cannulae</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7030&#160;</td>
			<td style="width:504px;">Guide cannulae implanted into brain region of interest to guide probe.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BASi cannula plastic headpiece for rats&#160;</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7011&#160;</td>
			<td style="width:504px;">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 ...

Research

JoVE Journal

Neuroscience

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...