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 溶液通过肾上腺静脉使用注射器和孵化组织20分钟在37°c。检查腺体以确保髓质的消化过程完成。组织应感到柔软而不紧张。</li>
	<li>收集经消化的髓质, 通过切断腺体的纵向方向。用手术刀将髓质组织剁碎。过滤组织悬浮在钢筛子和稀释以洛克的解答到大约50毫升容量减少胶原酶 P 的活动。</li>
	<li>在室温下将 300 x g 的细胞颗粒放入离心机中10分钟, 并将其收集成50毫升不育试管。在20毫升的溶液中重新悬浮获得的颗粒, 并将溶液在无菌100µm 尼龙网中过滤成管子。</li>
	<li>混合嗜细胞悬浮与无菌 Percoll (1:1) 和离心机的细胞溶液在 18600 x g 20 分钟室温。收集密度梯度的顶层, 并在100µm 尼龙网格上过滤溶液。</li>
	<li>要排除 Percoll, 请用洛克溶液稀释细胞悬浮液, 在室温下以 300 x g 为10分钟, 然后在洛克溶液中重新悬浮颗粒。分离细胞悬浮液的估计细胞密度约为400万细胞/毫升。</li>
	<li>对于胞吐作用和细胞内细胞仪测量的安培记录, 胶原蛋白 IV 的嗜细胞, 涂覆60毫米塑料碟, 密度约为 17.5 x10 3 细胞/厘米2, 在 37CO 5% 中孵化出2°c 的细胞。环境.在细胞培养1-3 天内进行电化学实验。</li>
	<li>对于 TEM 成像实验, 75 厘米2细胞培养皿中的板嗜细胞在每瓶7-8 元细胞的密度下, 在 5% CO2环境中孵育37°c 1 天。</li>
</ol>2. 单细胞胞吐作用探头实验24
<ol><li>为进行这些实验, 制备碳纤维电极, 取一个外径为电极的玻璃毛细管, 该外直径适用于这些实验所使用的磁头阶段。使用硼硅酸盐玻璃毛细管, 外径为1.2 毫米, 内径为0.69 毫米。<ol>
<li>将毛细管一端连接到吸水管。将5µm 直径的碳纤维放在某物上, 如一张白纸, 以增强可视性。</li>
		<li>确定一根碳纤维, 用手指将纤维放在一端, 保持碳纤维到位, 同时将毛细管的开口端定位到碳纤维的自由端附近。将碳纤维轻轻地吸进玻璃毛细管中, 使碳纤维通过毛细管两端伸出。移开吸入力。</li>
	</ol></li>
	<li>将毛细管与碳纤维放入微拉拔器中, 将玻璃毛细管拉出两个独立的玻璃吸管小贴士。要断开两个玻璃尖之间连接的碳纤维, 请使用剪刀切割碳纤维并获得两个碳纤维电极。</li>
	<li>在显微镜下, 将碳纤维放在较厚的显微镜滑梯上, 允许手工切割碳纤维的边缘, 纤维从玻璃毛细管涂层延伸到使用手术刀。</li>
	<li>切割碳纤维后, 留下一个玻璃涂层碳纤维尖端, 插入几毫米的电极尖端到环氧树脂溶液中10分钟拉起环氧树脂和密封任何可能的开放空间之间的碳纤维和周围的玻璃。将电极缓慢地从环氧树脂溶液中提起, 以防止在电极尖端形成粗大的胶滴。</li>
	<li>将电极放在持有者 (例如, 一个木制扁棍) 上, 用两条边粘着的耐热胶带将电极连接在一起。将环氧树脂处理过的电极在烤箱中隔夜烘烤100摄氏度。电极提示容易打破, 如果他们接触到表面, 所以确保电极始终是安全储存使用的持有人在那里电极固定到位, 并提示不冒险被触摸。</li>
	<li>要获得平面圆盘电极表面, 请将电极放在 microgrinder 的支架上, 并在45°的天使上将每个碳纤维电极斜角。在斜边后, 用一个永久性的标记标记其上表面的毛细管, 后来知道如何在将电极靠近电池胞吐作用测量时, 在45°角定位圆盘电极曲面。 注意: 这一步很重要, 因为在这些实验中, 椭圆圆盘电极需要放置在与培养皿表面平行的细胞顶部。此外, 斜边电极是在同一天进行的实验, 以确保一个新鲜和清洁电极表面。</li>
	<li>使用前, 将每个碳纤维微电极放在测试溶液中 (例如, PBS 缓冲器中的0.1 毫米多巴胺 (pH 7.4)) 以循环伏安法监测稳态电流。对于循环伏安扫描, 将三角形电位波形从-0.2 v 到 +0.8 V 与 Ag/AgCl 参考电极在 100 mV/秒中, 以确保良好的反应动力学数据得到与理论上计算的值为5µm 直径圆盘碳纤维微电极25。</li>
	<li>对于胞吐作用的探头记录, 将培养皿嗜细胞放到倒置显微镜上。重要的是要屏蔽与法拉第笼建立的显微镜, 以消除电子噪音在探头记录, 由于非常小的电流被测量。在细胞实验期间, 使用显微镜加热阶段保持37°c 的温度。</li>
	<li>使用低噪声膜片钳仪器, 在工作电极上应用 +700 mV 的恒定电位, 与在工作电极之外的培养皿中放置的 Ag/AgCl 参考电极。对于记录胞吐作用的嗜细胞, 数字化的信号在10赫和应用内部低通贝塞尔滤波器在2赫, 以过滤记录的信号。</li>
	<li>要在单个单元格上执行安培记录, 请在与恒电位仪一起使用的头部阶段的电极支架上安装新的斜面和经过测试的碳纤盘微电极。<ol>
<li>轻轻地将电极与扁椭圆形圆盘形状电极表面朝下, 朝向细胞的顶端表面, 然后用机器人将电极与细胞膜接触。</li>
		<li>通过监测电极在细胞膜顶部放置时所引起的细胞变形, 将电极与电极的距离进行调整, 然后仔细地将焊条缩回到一个距离, 使细胞恢复接近其原始细胞形状的形状。.理想的动力学和定量记录是创建一个100纳米薄液膜, 以分离电极和细胞表面, 这是类似的条件, 突触后检测的化学释放在突触。</li>
	</ol></li>
	<li>要刺激细胞胞吐作用, 请将玻璃微的顶端大小为2-3 µm 直径, 用5毫米BaCl 2 溶液在离细胞至少20µm 的距离内填充, 以防止从吸管尖端漏出的任何溶液影响实验和应用5秒的注入脉冲5毫米 BaCl2溶液在细胞表面刺激细胞胞吐作用。</li>
	<li>为了研究渗透压对胞吐作用过程的可逆影响, 准备一个等位缓冲液 (150 毫米氯化钠, 5 毫米氯化钾, 1.2 毫米 MgCl2, 5 毫米葡萄糖, 10 毫米 HEPES, pH 7.4) 与 310 mOsm/千克 iso 渗透压力和一个高渗缓冲由调整与 730 mOsm/千克对应的渗压缓冲液的 NaCl 浓度。</li>
	<li>将细胞放在等位缓冲中, 通过应用钡注入脉冲, 并在初始胞吐作用活动期间记录安培电流瞬变约3分钟, 刺激细胞胞吐作用。<ol>
<li>为了将等渗条件下的胞吐作用反应与高渗条件进行比较, 在高渗缓冲液中孵化出10分钟的细胞, 随后应用钡注入脉冲刺激胞吐作用, 执行3分钟安培记录。</li>
		<li>对于细胞的可逆反应, 在等渗缓冲液中再次孵化细胞10分钟, 通过应用5s 钡注入脉冲来刺激细胞, 并执行3分钟记录细胞胞吐作用反应。</li>
	</ol></li>
	<li>作为控制实验, 以确定对多钡刺激对胞吐作用活动的影响, 执行安培记录胞吐作用释放从三连续 BaCl 2 刺激对同一细胞放置在等渗缓冲和采用同一时间协议对不同渗透的连续细胞孵化实验。</li>
</ol>3. 细胞内电化学细胞术24,26
<ol><li>为了制造囊泡量子尺寸测量用电极, 使用相同的材料, 并开始准备5µm 直径碳纤维电极根据2.1 和2.2 节的说明, 电极制造的安培胞吐作用测量.</li>
	<li>在显微镜下, 使用手术刀切割从玻璃尖端延伸出来的碳纤维, 使从玻璃尖端伸出的碳纤维长度从30到100µm。</li>
	<li>要准备一个碳纤维电极的火焰蚀刻尖端, 使用丁烷火焰。要实现均匀蚀刻的圆柱形电极尖端, 请在旋转时按住圆柱形碳纤维电极, 将碳纤维从玻璃上延伸到丁烷火焰的蓝色边缘, 直到碳尖端形成红色。颜色。这通常需要少于2秒。如果成功, 这会导致蚀刻电极尖端的直径为 50-100 nm (如图 5B中所示的 SEM 图像)。火焰蚀刻后, 将电极置于显微镜下, 以评估电极尖端。</li>
	<li>将圆柱形 nanotip 微电极插入环氧树脂溶液中3分钟, 然后将电极尖端的十五年代浸渍到丙酮溶液中。这允许环氧树脂密封在碳纤维和中空玻璃毛细管壁之间的潜在间隙空间, 而丙酮清除环氧树脂的蚀刻碳纤维电极表面。要治疗环氧树脂, 在烤箱中烘烤电极在100摄氏度过夜。</li>
	<li>使用前, 测试每一个碳纤维微电极的稳态电流, 如2.7 节中使用循环伏安法所解释的。在实验中, 仅使用显示 1.5-2.5 nA27的高原电流的电极。</li>
	<li>对于细胞间探头测量, 将细胞置于显微镜下, 如2.8 所述, 并使用2.9 中所述的恒电位仪相同的实验设置。</li>
	<li>为了进行细胞内安培测量, 并防止对细胞造成重大的物理损伤, 通过添加一个温和的机械力, 将 nanotip 圆柱微电极插入细胞中, 刚好足以推动电极通过细胞等离子体膜和进入细胞胞浆使用机器人。</li>
	<li>插入后, 用细胞膜密封在圆柱电极周围, 开始在活细胞原位进行安培记录。在电极的氧化电位下, 囊泡会吸附到电极表面并随机破裂。 因此, 没有必要采取任何形式的刺激措施来启动这一进程。</li>
	<li>为了确定对囊泡量子大小的渗透效应, 从一组已在等渗和高渗缓冲中孵化出的细胞中收集到的胞内细胞术测量, 使用2.13 节所述的实验条件。</li>
</ol>4. 探头记录的数据分析
<ol><li>为了分析胞吐作用和胞内囊泡量子尺寸分析中记录的探头数据, 使用一个软件程序, 允许在记录的电流与时间轨迹上分析电流瞬态, 使运动峰值参数和可以确定单个电流峰值的综合总电荷。对于数据分析, 我们使用了在苏尔寿实验室中开发的用于数据分析程序28的软件。</li>
	<li>在分析安培峰值时, 选择每个记录的噪声的平均平方 (RMS) 标准偏差的峰值三倍的阈值限制。</li>
	<li>手动收集每个单元记录的安培峰值, 以防止不跟随高斯形状的假峰值, 可以是合并胞吐作用事件的结果的双峰值, 并且只接受具有阈值为三倍的高斯形峰值高于 RMS 噪声。</li>
	<li>收集每单安培峰值总电荷的数据, 并使用 Faradays 法 (n = QnF) 计算每胞吐作用或胞内单囊破裂事件中检测到的儿茶酚胺神经递质的摩尔量, 其中 N 是摩尔神经递质通过电极表面的氧化还原反应, Q = 每穗下检测到的总电荷, n = 在氧化还原反应中转移的电子数 (n=2 为儿茶酚胺) 和 Faraday´s 常数 F=96485 C/摩尔。</li>
	<li>对收集的数据进行统计分析, 首先计算每个单元每个事件检测到的平均电荷。然后, 对于细胞之间的差异, 计算细胞之间的平均电荷, 并使用学生的 t 测试之间的细胞假设不等方差。本研究采用 MATLAB 程序进行统计分析。</li>
</ol>5. TEM 成像对囊泡大小的分析
<ol><li>为了在不同的渗透条件下对细胞进行透射电镜成像, 首先在 5% CO2环境中, 在细胞化学固定前, 先在10分钟内将等位或高渗的细胞孵化为37摄氏度。</li>
	<li>使用 Karnovsky 固定方法29执行单元格定位。使用此方法, 用含有0.01% 叠氮化钠、1% 甲醛和1.25% 戊二醛的溶液孵育细胞, 样品可以储存在4摄氏度。</li>
	<li>固定后, 用0.15 米常使用钠缓冲细胞洗涤。</li>
	<li>为了染色细胞标本后固定, 并在准备 TEM 成像, 孵化细胞与1% 锇毒气溶液为2小时4摄氏度, 其次是 1 h 孵化在0.5% 的醋酸铀溶液在室温下在黑暗中。</li>
	<li>在最后的固定步骤中, 先用100% 乙醇冲洗细胞, 然后用丙酮冲洗细胞, 使细胞脱水。</li>
	<li>在环氧树脂中嵌入细胞样本, 然后以 4000 x g 为30-40 分钟进行离心, 并允许细胞样品在40摄氏度聚合为 15 h, 其次为48摄氏度孵化。</li>
	<li>在制备成像时, 将琼脂100树脂中嵌入的细胞样本分成 60 nm 的切片, 使用 ultramicrotome。</li>
	<li>将细胞以4% 铀 acetate/25%ethanol 溶液为4分钟, 然后用清水冲洗二十年代。用雷诺铅柠檬酸3分钟冲洗细胞, 然后用清水冲洗二十年代。</li>
	<li>对细胞样品进行透射电镜成像。在我们的实验中, 我们使用了一个透射电镜, 已经运行了120伏。</li>
	<li>从每一个细胞切片的记录图像中, 可以显示细胞胞浆中的囊泡的细胞超微结构, 首先要通过将像素与每个 TEM 图像中记录的刻度条相关, 来校准图像像素的大小。使用成像软件来确定每一个细胞在等渗或高渗条件下的图像中的囊泡大小。在实验中, 我们使用软件 ImageJ 进行图像分析。值得注意的是, 对于细胞的透射电镜图像进行细胞超微结构的图像分析, 这些测量的大小调整是基于细胞剖面的厚度需要考虑的, 以前是由 Almers´s 实验室30来描述的。</li>
</ol>

<ol>
	<li>Albillos, A., 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=The+exocytotic+event+in+chromaffin+cells+revealed+by+patch+amperometry.">The exocytotic event in chromaffin cells revealed by patch amperometry.</a> Nature. 389 (6650), 509-512 (1997).</li><li>Dunevall, 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=Characterizing+the+Catecholamine+Content+of+Single+Mammalian+Vesicles+by+Collision-Adsorption+Events+at+an+Electrode.">Characterizing the Catecholamine Content of Single Mammalian Vesicles by Collision-Adsorption Events at an Electrode.</a> J Am Chem Soc. 137 (13), 4344-4346 (2015).</li><li>Crivellato, E., Nico, B., Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromaffin+vesicle:+advances+in+understanding+the+composition+of+a+versatile,+multifunctional+secretory+organelle.">The chromaffin vesicle: advances in understanding the composition of a versatile, multifunctional secretory organelle.</a> Anat Rec. 291 (12), 1587-1602 (2008).</li><li>Morris, S., Schultens, H., Schober, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+osmometer+model+for+changes+in+the+buoyant+density+of+chromaffin+granules.">An osmometer model for changes in the buoyant density of chromaffin granules.</a> Biophys J. 20 (1), 33 (1977).</li><li>Winkler, H., Smith, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromaffin+granule+and+the+storage+of+catecholamines.">The chromaffin granule and the storage of catecholamines.</a> Handbook of Physiology. 6 (7), 321-399 (1975).</li><li>Phillips, J., Allison, Y., Morris, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+calcium,+magnesium,+copper+and+iron+in+the+bovine+adrenal+medulla.">The distribution of calcium, magnesium, copper and iron in the bovine adrenal medulla.</a> Neurosci. 2 (1), 147-152 (1977).</li><li>Est&#233;vez-Herrera, 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=ATP:+The+crucial+component+of+secretory+vesicles.">ATP: The crucial component of secretory vesicles.</a> Proc Natl Acad Sci. 113 (28), 4098-4106 (2016).</li><li>Machado, J. D., Camacho, M., Alvarez, J., Borges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+intravesicular+calcium+in+the+motion+and+exocytosis+of+secretory+organelles.">On the role of intravesicular calcium in the motion and exocytosis of secretory organelles.</a> Commun integr biol. 2 (2), 71-73 (2009).</li><li>Toll, L., Howard, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Mg2++ion-activated+ATPase+and+a+pH+gradient+in+the+storage+of+catecholamines+in+synaptic+vesicles.">Role of Mg2+ ion-activated ATPase and a pH gradient in the storage of catecholamines in synaptic vesicles.</a> Biochemistry. 17 (13), 2517-2523 (1978).</li><li>Terland, O., Flatmark, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbate+as+a+natural+constituent+of+chromaffin+granules+from+the+bovine+adrenal+medulla.">Ascorbate as a natural constituent of chromaffin granules from the bovine adrenal medulla.</a> FEBS letters. 59 (1), 52-56 (1975).</li><li>Camacho, M., Machado, J. D., Montesinos, M. S., Criado, M., Borges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intragranular+pH+rapidly+modulates+exocytosis+in+adrenal+chromaffin+cells.">Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells.</a> J Neurochem. 96 (2), 324-334 (2006).</li><li>Jankowski, J. A., Schroeder, T. J., Ciolkowski, E. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+characteristics+of+quantal+secretion+of+catecholamines+from+adrenal+medullary+cells.">Temporal characteristics of quantal secretion of catecholamines from adrenal medullary cells.</a> J Biol Chem. 268 (20), 14694-14700 (1993).</li><li>Daniels, A., Williams, R., Wright, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+character+of+the+stored+molecules+in+chromaffin+granules+of+the+adrenal+medulla:+a+nuclear+magnetic+resonance+study.">The character of the stored molecules in chromaffin granules of the adrenal medulla: a nuclear magnetic resonance study.</a> Neurosci. 3 (6), 573-585 (1978).</li><li>Borges, R., Travis, E. R., Hochstetler, S. E., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+external+osmotic+pressure+on+vesicular+secretion+from+bovine+adrenal+medullary+cells.">Effects of external osmotic pressure on vesicular secretion from bovine adrenal medullary cells.</a> J Biol Chem. 272 (13), 8325-8331 (1997).</li><li>Troyer, K. P., Mundorf, M. L., Fries, H. E., Wightman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+vesicle+fusion+and+exocytosis+in+hypertonic+conditions.">Separating vesicle fusion and exocytosis in hypertonic conditions.</a> Ann NY Acad Sci. 971 (1), 251-253 (2002).</li><li>Brodwick, M. S., Curran, M., Edwards, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+osmotic+stress+on+mast+cell+vesicles+of+the+beige+mouse.">Effects of osmotic stress on mast cell vesicles of the beige mouse.</a> J Membrane Biol. 126 (2), 159-169 (1992).</li><li>Amatore, C., Arbault, S., Bonifas, I., Lemaitre, F., Verchier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+exocytosis+under+hypotonic+conditions+shows+two+distinct+populations+of+dense+core+vesicles+in+bovine+chromaffin+cells.">Vesicular exocytosis under hypotonic conditions shows two distinct populations of dense core vesicles in bovine chromaffin cells.</a> Chem Phys Chem. 8 (4), 578-585 (2007).</li><li>Hampton, R. Y., Holz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+changes+in+osmolality+on+the+stability+and+function+of+cultured+chromaffin+cells+and+the+possible+role+of+osmotic+forces+in+exocytosis.">Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis.</a> J Cell Biol. 96 (4), 1082-1088 (1983).</li><li>Troyer, K. P., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+separation+of+vesicle+release+from+vesicle+fusion+during+exocytosis.">Temporal separation of vesicle release from vesicle fusion during exocytosis.</a> J Biol Chem. 277 (32), 29101-29107 (2002).</li><li>Pihel, K., Travis, E. R., Borges, R., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocytotic+release+from+individual+granules+exhibits+similar+properties+at+mast+and+chromaffin+cells.">Exocytotic release from individual granules exhibits similar properties at mast and chromaffin cells.</a> Biophys J. 71 (3), 1633 (1996).</li><li>Jankowski, J. A., Finnegan, J. M., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ionic+composition+alters+kinetics+of+vesicular+release+of+catecholamines+and+quantal+size+during+exocytosis+at+adrenal+medullary+cells.">Extracellular ionic composition alters kinetics of vesicular release of catecholamines and quantal size during exocytosis at adrenal medullary cells.</a> J Neurochem. 63 (5), 1739-1747 (1994).</li><li>Leszczyszyn, D. 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. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+osmotic+stress+reduces+the+vesicle+size+while+keeping+a+constant+neurotransmitter+concentration.">Extracellular osmotic stress reduces the vesicle size while keeping a constant neurotransmitter concentration.</a> ACS Chemical Neuroscience. , (2017).</li><li>Bath, B. 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=Subsecond+adsorption+and+desorption+of+dopamine+at+carbon-fiber+microelectrodes.">Subsecond adsorption and desorption of dopamine at carbon-fiber microelectrodes.</a> Anal Chem. 72 (24), 5994-6002 (2000).</li><li>Li, X., Majdi, S., Dunevall, J., Fathali, H., Ewing, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Measurement+of+Transmitters+in+Individual+Vesicles+in+the+Cytoplasm+of+Single+Cells+with+Nanotip+Electrodes.">Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes.</a> Angew Chem Int Edit. 54 (41), 11978-11982 (2015).</li><li>Zoski, C. G., Mirkin, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+limiting+currents+at+finite+conical+microelectrodes.">Steady-state limiting currents at finite conical microelectrodes.</a> Anal Chem. 74 (9), 1986-1992 (2002).</li><li>Mosharov, E. V., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+exocytotic+events+recorded+by+amperometry.">Analysis of exocytotic events recorded by amperometry.</a> Nat Methods. 2 (9), 651-658 (2005).</li><li>Morris, 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+formaldehyde+glutaraldehyde+fixative+of+high+osmolality+for+use+in+electron+microscopy.">A formaldehyde glutaraldehyde fixative of high osmolality for use in electron microscopy.</a> J Cell Biol. 27, 137-139 (1965).</li><li>Parsons, T. D., Coorssen, J., Horstmann, H., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Docked+granules,+the+exocytic+burst,+and+the+need+for+ATP+hydrolysis+in+endocrine+cells.">Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells.</a> Neuron. 15 (5), 1085-1096 (1995).</li></ol>

作者没有什么可透露的。

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 ...

monitoring-effect-osmotic-stress-on-secretory-vesicles

Research

JoVE Journal

Neuroscience

8.4K 次观看.  Chalmers University of Technology. 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。

视频: Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

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

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

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 Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

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

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

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

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

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

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

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

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 of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

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

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

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 ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

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

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

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 ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

基于旋转加速大鼠漫射性无羟基脑损伤的诱导

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

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

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the &#945;3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the &#945;3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

We provide a protocol to generate a pharmacological DYT/PARK-ATP1A3 dystonia mouse model via implantation of cannulas into basal ganglia and cerebellum connected to osmotic pumps. We describe the induction of dystonia-like movements via application of a motor challenge and the characterization of the phenotype via behavioral scoring systems.

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 ...