Name: monitoring the effect of osmotic stress on secretory vesicles and exocytosis
Uploaded: 2018-02-19T13:30:00
Description: Watch this Scientific Journal Video about Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis at JoVE.com

作者感谢瑞典研究理事会 (349-2007-8680) 资助和 Dalsjöfors Kött AB (瑞典 Dalsjöfors) 捐赠牛肾上腺。

1. 肾上腺酶消化分离牛嗜细胞的细胞培养
<ol><li>为了分离从牛肾上腺收集的嗜细胞, 用70% 乙醇溶液消毒细胞。用手术刀修剪脂肪和结缔组织。除去血液, 用洛克的溶液 (氯化钠154毫米, 氯化钾5.6 毫米, NaHCO3 3.6 毫米, 羟乙基 piperazineethanesulfonic 酸 (HEPES) 5.6 毫米在 pH 7.4) 冲洗肾上腺静脉自动温控到37摄氏度。</li>
	<li>要消化组织, 充气每个腺体通过注射大约2.5 毫升温暖无菌过滤0.2% 胶原酶 P 溶液通过肾?...

<ol>
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J., 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=Nicotinic+receptor-mediated+catecholamine+secretion+from+individual+chromaffin+cells.+Chemical+evidence+for+exocytosis.">Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells. Chemical evidence for exocytosis.</a> J Biol Chem. 265 (25), 14736-14737 (1990).</li><li>Wightman, R., 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=Temporally+resolved+catecholamine+spikes+correspond+to+single+vesicle+release+from+individual+chromaffin+cells.">Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.</a> P Natl Acad Sci. 88 (23), 10754-10758 (1991).</li><li>Fathali, H., Dunevall, J., Majdi, S., Cans, A. -. 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我们这里提出了一个协议和三互补分析方法结合分析分泌性囊泡和胞吐作用过程的优点, 以更好地了解渗透压等物理力如何影响分泌性囊泡分泌细胞中的胞吐作用过程。这些方法包括碳纤维微电极探头, 它是一种记录胞吐作用活性的建立方法, 细胞内电化学细胞术, 用于确定量子在其当地环境中的囊泡大小, 并透射电镜图像分析监测分泌囊泡体积。在这项工作中, 通过收集探头记录的定量数据, 在暴?...

肾上腺中的嗜细胞是神经内分泌细胞, 释放神经递质分子进入血流。这是通过一个细胞过程, 其中包括对接和融合的神经递质囊泡, 导致内容释放从囊泡到细胞外空间的过程称为胞吐作用。嗜细胞中的神经递质 (肾上腺素和去甲肾上腺素) 被膜蛋白主动输送到大致密核囊泡 (LDCVs) 中, 并存储在高浓度 (~ 0.5-1 米)1,2。LDCVs 内神经递质的积累是由儿茶酚胺分子与由 chromogranin 蛋白 (169 毫克/毫升)3,4,5 组成的 intravesicular 致密核心蛋白基质的亲和性完成的. 、6和一个 intravesicular 鸡尾酒解决方案, 其中包含用于儿茶酚胺加载和存储的关键组件, 如 ATP (125-300 mM)7、Ca2 + (解决方案中的50-100 µM 和 ~ 40 毫米绑定到蛋白质矩阵)8, Mg2 + (5 毫米)9, 抗坏血酸 (10-30 毫米)10, pH 值为 ~ 5.511,12。LDCVs 保持一个 iso 渗透条件与细胞细胞质 (310 mOsm/千克)13, 即使理论溶质浓度在囊泡总和高达750毫米。intravesicular 组分的组成不仅对儿茶酚胺的加载和贮存至关重要, 而且对溶质聚集到致密的核心蛋白基质中也是必不可少的。这大大减少了囊泡的渗透, 并可能影响在胞吐作用5, 6 中释放的儿茶酚胺数量.
通过安培记录对细胞外渗透压对胞吐作用过程的影响的研究报告说, 高胞外渗透压抑制胞吐作用活性, 并减少从单一的神经递质分泌的数量囊泡间隔4,14,15,16,17,18,19。对这些观察的解释推测了在细胞胞浆抑制囊泡融合事件中大分子拥挤的可能增强, 以及膜生物物理特性的改变影响了神经递质在胞吐作用期间释放。这些想法假设高胞外渗透应力不影响囊泡量子大小, 这定义了在触发胞吐作用的前一阶段囊泡室中储存的神经递质分子的数量14,15,17,19,20,21. 在单细胞胞吐作用释放量的安培测量中, 碳纤盘微电极与细胞表面紧密接触, 形成模拟突触构型的实验设置, 其中安培电极作为突触后检测器 (图 1)22,23。通过刺激细胞胞吐作用, 可以诱导神经递质囊泡与细胞膜融合, 释放部分或全部囊泡含量进入细胞外空间。在电极表面释放的这些神经递质分子可以电化学检测, 如果神经递质是活性 (例如, 儿茶酚胺) 通过应用氧化还原电位 +700 mV vs 银/AgCl 参考电极.因此, 一系列的电流峰值标志着对个别胞吐作用事件的检测。从安培记录中的电流与时间轨迹, 每一个安培峰值下的区域表示每胞吐作用事件检测到的电荷, 并且可以转换为释放的神经递质的摩尔, 使用法拉第方程。因此, 安培记录提供定量信息的数量的神经递质从单一的胞吐作用事件和报告的频率胞吐作用事件, 但不提供定量信息的分泌性囊泡在囊泡融合和神经递质释放之前的内容已经开始。
因此, 为了更好地理解细胞细胞质分泌囊在细胞被触发接受胞吐作用前对细胞外渗透应力的反应, 其他互补分析方法可以用来丰富这些信息。例如, 研究渗透应力是否改变囊泡体积, 透射电镜 (TEM) 成像分析可用于测定化学固定后细胞的囊泡大小。为探讨渗透胁迫对囊泡量子大小的影响, 一种新近开发的称为胞内电化学细胞术的安培技术, 可用于在分泌性囊泡中定量测定囊泡神经递质含量。在活细胞的细胞质中仍然居住的本机状态26。在细胞内电化学细胞技术中, nanotip 柱状碳纤维电极轻轻地插入活细胞的细胞质中, 并将 +700 mV 电位应用于该电极 (vs a Ag/AgCl 参考电极),在囊泡中儿茶酚胺含量可以通过检测单个囊泡的氧化还原电流峰值来进行量化, 然后在电极表面进行随机破裂, 从而释放出它们的内容。电极表面26。因此, 在电流与时间的跟踪中, 每一个囊泡破裂事件都可能导致电流瞬变, 通过整合每个电流穗的面积, 可以用 Faraday´s 法计算囊泡量子大小。
因此, 通过将囊泡尺寸测量所得的定量信息与囊泡量子大小分析相结合, 经胞内电化学细胞仪记录, 囊泡神经递质浓度也可被确定。这允许囊泡的特征, 当细胞暴露于不同的渗透条件, 并提供了一个更好的看法, 如何水泡可能对细胞外渗透应力反应在胞吐作用之前的阶段。结合这些方法的结果表明, 在胞外高渗透压的存在下, 囊泡收缩和调整其量子大小, 并比较这些测量的相对变化的定量信息表明,收缩时, 囊泡调整其含量和大小以维持恒定的神经递质浓度24。因此, 这种理解对于连接到渗透胁迫下细胞中神经递质释放的观察是有价值的。在这些协议中, 我们描述了使用这三互补方法, 允许描述在他们的家乡环境中分泌的囊泡对细胞外渗透的反应以及这种反应对胞吐作用的影响。过程.除了我们以前关于高渗透压对胞吐作用24 影响的观察外, 我们还提出了进一步的实验, 描述渗透休克后细胞的恢复和多钡刺激在嗜中的作用。细胞.

<table border="0" cellpadding="0" cellspacing="0" style="width:631px;" width="631">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">NaCl</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">KCl</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">NaHCO3</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">HEPES</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">MgCl2</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">M-2670</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glucose</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Collagenase P</td>
			<td style="width:129px;">Roche, Sweden</td>
			<td style="width:119px;">11 213 857 001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">100-&#181;M Nylon mesh</td>
			<td style="width:129px;">Fisher Sientific</td>
			<td style="width:119px;">08-771-19</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Percoll</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">P1677</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Collagen IV coated 60 mm plastic dish</td>
			<td style="width:129px;">VWR</td>
			<td style="width:119px;">354416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Centrifuge</td>
			<td style="width:129px;">Avanti J-20XP</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Borosilicate glass capillary</td>
			<td style="width:129px;">Sutter instrument Co., Novato, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Micropipette puller</td>
			<td style="width:129px;">Narishing Inc., Japan</td>
			<td style="width:119px;">PE-21</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Epoxy solutions (A and B)</td>
			<td style="width:129px;">Epoxy technology, Billerica, MA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Beveller</td>
			<td style="width:129px;">Narishing Inc.</td>
			<td style="width:119px;">EG-400</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Inverted Microscope</td>
			<td style="width:129px;">Olympus</td>
			<td style="width:119px;">IX81</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Patch clamp Instrument</td>
			<td style="width:129px;">Molecular Devices, Sunnyvale, CA</td>
			<td style="width:119px;">Axopatch 200B</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Micromanipulator</td>
			<td style="width:129px;">Burleigh Instrument Inc., USA</td>
			<td style="width:119px;">PCS-5000</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Butane Flame</td>
			<td style="width:129px;">Multiflame AB, H&#228;sselholm, Sweden</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission electron microscopy</td>
			<td style="width:129px;">Omega</td>
			<td style="width:119px;">Leo 912 AB</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring the effect of osmotic stress on secretory vesicles and exocytosis

探头对渗透休克细胞的记录表明, 分泌细胞通过减少单个胞吐作用事件中的胞吐作用活性和囊泡释放的神经递质的数量来应对这一生理压力。有人建议, 减少神经递质的降低是由于细胞膜生物物理性质的变化, 当细胞收缩反应渗透胁迫, 并假设, 分泌泡在细胞胞浆不受影响细胞外渗透应力。探头记录胞吐作用监视器当囊泡与等离子膜融合时, 从细胞中释放出的是什么, 但在触发囊泡聚变之前没有提供有关囊泡含量的信息。因此, 通过结合探头记录与其他补充分析方法, 能够表征分泌囊泡在胞吐作用细胞被触发提供了一个更广泛的概述, 以检查如何分泌囊泡和胞吐作用过程受渗透冲击的影响。我们在这里描述如何补充探头记录与细胞内电化学细胞术和透射电镜 (TEM) 成像可以用来表征变化的分泌囊泡大小和神经递质的内容在嗜细胞与胞吐作用活性有关, 暴露于渗透胁迫前后。通过将从实验中获得的定量信息与所有三种分析方法联系起来, 得出结论: 分泌性囊泡通过缩小大小和减少囊泡量子大小来响应胞外渗透应力, 以保持一个恒定的囊泡神经递质浓度。因此, 这给出了一些解释, 为什么囊泡, 响应渗透应激, 减少释放的神经递质胞吐作用释放。这里的安培记录表明这是一个可逆的过程, 当被放置的细胞恢复到一个等质环境中时, 渗透休克后的囊泡会重新注入神经传递素。

我们在这里描述了如何将 TEM 成像与两种电化学方法、碳纤维探头和细胞内电化学细胞术相结合的协议, 可以提供信息, 得到更广泛的观点, 暗指的效果分泌囊泡的胞外渗透压和胞吐作用过程。通过在单个嗜单元格中使用实验设置 (如图 1所示) 比较胞吐作用释放的代表安培记录, 当细胞接触渗透时, 显示 exocytotic 活动显著减少。与等渗条件下的单元格相比的应力 (<strong class="xfig"...

Watch this Scientific Journal Video about Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis at JoVE.com

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

渗透胁迫影响胞吐作用和在这个过程中释放的神经递质的数量。我们演示了如何结合电化学方法和透射电子显微术来研究细胞外渗透压对胞吐作用活性、囊泡量子大小和释放神经递质的影响。在胞吐作用期间。

渗透胁迫对分泌性囊泡和胞吐作用的监测作用

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Research

JoVE Journal

Neuroscience

Low-stress Route Learning Using the Lashley III Maze in Mice

低应力路线学习，在老鼠身上使用拉什利三迷宫

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

科研

神经科学

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

脑成像的情感记忆的促进作用调查

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the ...

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

脑成像的情感对认知损害的影响调查

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

使用扩频萧条的情节和慢性偏头痛的神经免疫信号建模<em在体外</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

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 ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

免疫细胞衍生细胞外囊泡的表征及研究功能对细胞环境的影响

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

渗透泵的植入和压力的诱导，以建立一个症状，药理学小鼠模型DYT/PARK-ATP1A3肌张

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do ...

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

基于渗透泵的药物递送用于中枢神经系统 体内 髓鞘再生研究

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and ...