Name: combined infusion and stimulation with fast-scan cyclic voltammetry (cis-fscv) to assess ventral tegmental area receptor regulation of phasic dopamine
Uploaded: 2020-04-23T14:00:00.000+00:00
Duration: 6 min 40 s
Description: Watch this Scientific Journal Video about Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine at JoVE.c

工作得到了伊丽莎白镇学院（R.J.W，M.L.和L.M），NSF研究生奖学金（R.J.W.）和耶鲁大学医学院（N.A.）的支持。

所有实验均根据美国国立卫生研究院（NIH）实验动物护理和使用指南进行，并得到伊丽莎白镇学院和耶鲁大学机构动物护理和使用委员会（IACUC）的批准。该协议特定于利用CIS-FSCV的麻醉大鼠制剂。
1. 术前准备
<ol><li>电极溶液制备<ol>
<li>为了使电极回填溶液，制备4M醋酸钾与140mM氯化钾16的溶液。</li>
	</ol>
</li>
	<li>电极制备<ol>
<li>使用真空抽吸，将T-650碳纤维（直径7μm）插入硼硅酸盐玻璃毛细管（长度= 100 mm，直径= 1.0 mm，内径= 0.5 mm）。</li>
		<li>一旦碳纤维被放置在玻璃毛细管内，将玻璃毛细管放入垂直电极拉拔器中，热元件大致位于毛细管的中间。在磁铁关闭的情况下，将加热器设置为 55。</li>
		<li>拉动毛细管后，小心地抬起上部毛细管支架，使电极的尖端不被加热元件包围。</li>
		<li>使用锋利的剪刀，切掉仍然连接毛细管两块的碳纤维。这将导致两个独立的碳纤维微电极。</li>
		<li>在光学显微镜下，用锋利的手术刀小心地切割暴露的碳纤维，使碳纤维延伸到玻璃末端外约75-100μm。</li>
		<li>使用光学显微镜，确保电极沿毛细管没有裂缝。还要确保碳纤维离开毛细管的密封件难以注意到并且没有裂缝。 注意:良好的密封有助于减少录制过程中的噪音。参见已发表的研究17，18，19以获取更详细的方案。</li>
	</ol>
</li>
	<li>参比电极制造<ol>
<li>将金针焊接到5厘米银线上。</li>
		<li>将阳极连接到金属回形针或其他导体上，将阴极连接到引脚上，并在回形针和银线浸没在0.1 M HCl中时施加电压（~2 V）。</li>
		<li>一旦银线上出现白色涂层（AgCl），停止电压。</li>
	</ol>
</li>
	<li>准备用于植入的电极<ol>
<li>将金针焊接到一根细绝缘线（长约10厘米，直径<0.50毫米）上。</li>
		<li>从与金针相对的电线上取下约5厘米的绝缘层。</li>
		<li>用电极溶液填充电极大约一半。</li>
		<li>将绝缘线插入电极。 注:导线应与电极内部的碳纤维接触。</li>
	</ol>
</li>
</ol>2. 电极植入
<ol><li>给予成年，雄性，Sprague Dawley大鼠（250-450g）腹膜内注射（1.5g / kg或1 mL / kg体积）0.5g / mL氨基甲酸酯溶解在无菌盐水中。从初始聚氨酯剂量1.0-1.2克/千克开始。如果动物在氨基甲酸酯给药20分钟后仍然对有毒刺激试验（尾部捏）有反应，则额外给予0.3-0.5g / kg聚氨酯，总剂量为1.5g / kg。 注意:为了制备0.5g / mL聚氨酯溶液，将10g氨基甲酸酯加入10g（~10mL）盐水中。聚氨酯是一种致癌物质，必须小心处理。聚氨酯是一种重要的麻醉剂，因为它不会改变多巴胺的水平，就像其他麻醉剂如氯胺酮/木肼嗪和水合氯醛20，21一样。</li>
	<li>一旦动物被深度麻醉并且对有害刺激（例如，脚趾捏）没有反应，将其置于立体定位框架中。将眼科润滑剂涂抹在大鼠的每只眼睛上。 注意:这是一种非生存手术，但鼓励使用良好的无菌技术。</li>
	<li>使用两阶段磨砂膏清洁大鼠的头皮（即，碘泊维酮磨砂膏，然后进行70%乙醇磨砂;重复3个周期）。</li>
	<li>使用无菌针鼻镊子和手术剪刀切除头皮组织。去除大量组织，为下面列出的各种植入留出空间。</li>
	<li>使用无菌的棉质尖端涂抹器轻轻清洁颅骨表面。然后滴2-3滴3%的过氧化氢，以帮助识别λ和bregma。</li>
	<li>使用立体定位或手钻（1.0 mm，~20，000 rpm），钻一个直径1.5 mm的孔，前部2.5 mm，侧钻3.5 mm的孔。部分（大约一半，直到它牢固地到位）将螺钉（1.59 mm外径，3.2 mm长）植入该孔中。建议在钻孔时使用无菌盐水灌溉，以防止热损伤。</li>
	<li>对于参比电极，在左半球钻一个直径为1.0 mm的孔，前部1.5 mm，侧孔为3.5 mm。</li>
	<li>用手将~2 mm的参考线插入该孔中，同时将参考线缠绕在植入螺钉的头部周围和下方。</li>
	<li>完全植入螺钉，将参比电极固定到位。</li>
	<li>在右半球，钻一个直径1.5毫米的孔，前孔1.2毫米，侧钻1.4毫米。</li>
	<li>用镊子轻轻取下硬脑膜。</li>
	<li>对于刺激电极，钻一个方孔（前后2 mm，内侧5 mm），中心位于后方5.2 mm，侧向1.0 mm。</li>
	<li>使用立体定向臂杆，将双极刺激电极/导套管降低到硬脑膜下方5 mm处。如果在电极植入过程中出血，请使用无菌棉签和纱布以尽量减少出血。 注:本方法中使用的双极刺激电极预先装有导引套管（材料表）。与本产品一起使用的内部套管在完全插入导向套管时，应用双极刺激电极上的插脚冲洗。这将允许内部套管直接位于刺激器的两个插脚之间，它们相距约1毫米。类似的协议在14的其他地方进行了描述。</li>
	<li>使用立体定向臂杆，将碳纤维微电极降低到硬脑膜以下4毫米处。这个位置位于纹状体的最背侧部分。</li>
	<li>将参考线和碳纤维连接到恒电位仪。</li>
	<li>在60 Hz下施加三角波形式（-0.4−1.3 V，400 V / s）15分钟，并在10 Hz下再次施加10分钟。 注意:通常，当将波形施加到大脑中的碳纤维微电极上时，氧化基团被添加到碳纤维的表面。在记录之前必须达到该反应的平衡;否则将发生显著漂移19。以更高的频率（60 Hz）循环电极可以使碳纤维更快地达到平衡。</li>
</ol>3. 优化碳纤维和刺激电极/导套管位置
<ol><li>将刺激器设置为产生双极性电波形，频率为 60 Hz，24 个脉冲，300 μA 电流，脉冲宽度为 2 ms/相。</li>
	<li>以0.2 mm的增量将刺激器从硬膜下方的5 mm降低到7.8 mm。在每个增量中，刺激 VTA。 注意:在更深的背深（5-6毫米），大脑的刺激通常会（约80%的时间）导致大鼠的胡须抽搐。在更深的深度，晶须将停止抽搐，这发生在硬脑膜下方7.5-8.2毫米之间。当晶须停止抽搐时，刺激电极将靠近或位于VTA。这不会发生在每只大鼠身上，并且不应将缺乏胡须抽搐视为双极刺激电极/输液套管错位的迹象。并非所有麻醉剂（例如异氟醚）都可能发生晶须抽搐。</li>
	<li>继续降低双极刺激电极/导套管，直到刺激在碳纤维微电极（目前在背纹状体）处产生相位DA释放。 注意:如果双极电极植入VTA中，则背纹状体中的DA释放并不总是发生，但是在VTA刺激下观察到背纹状体中的DA释放通常是一个好迹象，表明在NAc核心中会观察到良好的信号。</li>
	<li>降低碳纤维微电极，直到它至少低于硬脑膜6.0毫米。这是NAc核心的最背侧部分。</li>
	<li>刺激VTA并记录DA峰值的峰值幅度。</li>
	<li>降低或升高产生最大DA释放的位点的碳纤维微电极。</li>
	<li>确保DA响应的峰值是0.6 V时的清晰氧化峰和-0.2 V时的还原峰。这些峰值表示 DA。</li>
</ol>4. 联合输注和刺激 FSCV 记录
注: 图1 显示了VTA微输注前后记录的时间线。
<ol><li>一旦碳纤维和刺激电极/导套管位置得到优化，刺激约20-30分钟。 注意:在电流刺激参数下，每3分钟刺激一次不要超过一次，以允许囊泡重新加载22。</li>
	<li>在达到稳定的基线（五次刺激<20%的变化）后，用手将内部套管轻轻降低到预先安装到双极刺激器中的导套管中。</li>
	<li>再进行2-3次基线记录，以确保插管插入本身不会导致诱发信号发生变化。在某些情况下，插入和拔出内部套管可能会对VTA造成损坏。如果信号在此基线周期内发生剧烈变化（>20%），则再进行3-4次记录，直到基线重新稳定。</li>
	<li>使用注射器泵和微量注射器，在2分钟内将0.5μL溶液（例如，0.9%盐水，N-甲基-D-天冬氨酸[NMDA]，（2R）-氨基-5-膦源性戊酸[AP5]）注入VTA中。</li>
	<li>输注后，在取出前将内套管放置至少1分钟。 注意:根据药物动力学，某些药物可能需要将内部套管放置更长时间，并且移除内部套管可能会导致药物通过内部套管返回。如果有顾虑，可以在整个录制过程中将内部套管留在引导套管中。否则，可以在此 1 分钟间隔后开始录制。</li>
	<li>继续每3分钟记录一次，以测量输注后效果。 注:如果输注对照溶液，但未观察到任何效果，则可第二次输注10。如果插入内插管或盐水输注导致DA释放改变，则信号通常在30分钟内恢复到基线。</li>
</ol>5. 电极放置的组织学验证
<ol><li>在实验结束时，使用碳纤维微电极在记录部位创建一个小病变。<ol>
<li>如果必须保留电极以进行实验后校准，则使用钨丝放置在毛细管尖端外约100μm的玻璃毛细管中。在这种情况下，从大脑抬起电极，用钨电极代替记录电极，并将其降低到相同的背侧坐标。 注意:碳纤维也可用于损伤大脑，并将更准确地表示记录部位的位置;但是，实验者将失去校准这些电极的能力。</li>
	</ol>
</li>
	<li>要损伤记录部位，请使用电源施加电压。从1 V开始，每10秒增加1 V，直到达到10 V。</li>
	<li>使用致命的腹膜内注射戊巴比妥（150mg / kg）对动物实施安乐死。</li>
	<li>使用4%福尔马林溶液灌注大鼠。</li>
	<li>使用削尖的断头台将头部从老鼠身上取下。</li>
	<li>使用rongeurs，去除大脑周围的结缔组织和颅骨，并轻轻地将大脑从任何剩余的组织中移开。</li>
	<li>将大脑储存在4%福尔马林中1天，然后将其转移到30%蔗糖中。 注意:用4%福尔马林灌注不需要看到病变部位，尽管作为最佳实践，它将改善病变部位的重建。</li>
	<li>使用低温恒温器创建30μm的大脑切片。</li>
	<li>将切片安装在载玻片上，并用盖玻片盖住。</li>
	<li>表示碳纤维微电极病变的位置和使用光学显微镜的双极刺激器/输注套管的位置。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast-scan+cyclic+voltammetry+of+5-hydroxytryptamine.">Fast-scan cyclic voltammetry of 5-hydroxytryptamine.</a> Analytical Chemistry. 67 (6), 1115-1120 (1995).</li><li>Park, J., Takmakov, 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=In+vivo+comparison+of+norepinephrine+and+dopamine+release+in+rat+brain+by+simultaneous+measurements+with+fast-scan+cyclic+voltammetry.">In vivo comparison of norepinephrine and dopamine release in rat brain by simultaneous measurements with fast-scan cyclic voltammetry.</a> Journal of Neurochemistry. 119 (5), 932-944 (2011).</li><li>Wenzel, J. M., 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=Phasic+Dopamine+Signals+in+the+Nucleus+Accumbens+that+Cause+Active+Avoidance+Require+Endocannabinoid+Mobilization+in+the+Midbrain.">Phasic Dopamine Signals in the Nucleus Accumbens that Cause Active Avoidance Require Endocannabinoid Mobilization in the Midbrain.</a> Current Biology. 28 (9), 1392-1404 (2018).</li><li>Spanos, M., 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=NMDA+Receptor-Dependent+Cholinergic+Modulation+of+Mesolimbic+Dopamine+Cell+Bodies:+Neurochemical+and+Behavioral+Studies.">NMDA Receptor-Dependent Cholinergic Modulation of Mesolimbic Dopamine Cell Bodies: Neurochemical and Behavioral Studies.</a> ACS Chemical Neuroscience. 10 (3), 1497-1505 (2019).</li><li>Cheer, J. F., 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=Cannabinoids+enhance+subsecond+dopamine+release+in+the+nucleus+accumbens+of+awake+rats.">Cannabinoids enhance subsecond dopamine release in the nucleus accumbens of awake rats.</a> Journal of Neuroscience. 24 (18), 4393-4400 (2004).</li><li>Melchior, J. 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=Optogenetic+versus+electrical+stimulation+of+dopamine+terminals+in+the+nucleus+accumbens+reveals+local+modulation+of+presynaptic+release.">Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release.</a> Journal of Neurochemistry. 134 (5), 833-844 (2015).</li><li>Sun, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genetically+Encoded+Fluorescent+Sensor+Enables+Rapid+and+Specific+Detection+of+Dopamine+in+Flies,+Fish,+and+Mice.">A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice.</a> Cell. 174 (2), 481-496 (2018).</li><li>Robinson, D. L., 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=Monitoring+rapid+chemical+communication+in+the+brain.">Monitoring rapid chemical communication in the brain.</a> Chemical Reviews. 108 (7), 2554-2584 (2008).</li><li>Park, 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=Heterogeneous+extracellular+dopamine+regulation+in+the+subregions+of+the+olfactory+tubercle.">Heterogeneous extracellular dopamine regulation in the subregions of the olfactory tubercle.</a> Journal of Neurochemistry. 142 (3), 365-377 (2017).</li><li>Ganesana, M., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+changes+in+transient+adenosine+during+cerebral+ischemia+and+reperfusion+injury.">Early changes in transient adenosine during cerebral ischemia and reperfusion injury.</a> PLoS One. 13 (5), e0196932 (2018).</li></ol>

作者没有什么可透露的。

CIS-FSCV为研究相位DA释放的潜在VTA受体机制提供了独特的机会。为了确保正确录制，有两个关键步骤。首先，必须实现稳定的基线记录，并且诱发的DA信号漂移很小。增加建立稳定记录的可能性的一个重要方法是确保电极有足够的时间在60 Hz和10 Hz下循环（通常在60 Hz下为15分钟，在10 Hz下为10分钟）。当碳纤维被循环时，碳纤维本身氧化并变得蚀刻，减少表面积，但产生用于多巴胺吸附的新表面23。这可能导致对DA的敏感性增加。因此，随着时间的推移，人们可能会看到实验中刺激的多巴胺释放略有增加，这是由于这种增加的蚀刻，而不是任何药理学操作。此外，在初始麻醉后 90 至 120 分钟内开始记录将增加长时间稳定记录的可能性。因此，当大鼠因麻醉而濒临死亡时，诱发的DA释放通常会缓慢减少。
该过程的第二个关键步骤是确保输注套管轻轻插入双极刺激电极中。如果在插入内部套管时施加太大的压力，立体定位臂杆可以移动，因此，多巴胺信号可能会人为地增加或减少，因为刺激部位可能不同。如果插管插入后诱发信号发生显著变化，应建立新的基线周期。此外，如果插入内部套管或载体输注导致DA释放改变，则信号通常在30分钟内恢复到基线。如果对载体输注的DA释放发生广泛改变，输注速率或体积可能会降低。研究人员还可以在输注前插入内部套管后进行额外的记录，以评估插管本身的插入是否可以改变释放。与此相关的是，重要的是要验证输注是否发生，并且没有阻塞内部套管。一种方法是在输液管中形成一个小气泡，并用笔或记号笔标记出来。输注后气泡应远离标记物。确保输注正确发生的另一种方法是在从大脑中取出内部套管后打开输液泵，如果尖端仍有溶液形成，则可能发生成功的输注。
CIS-FSCV可以适应于研究行为幼稚和训练有素的动物的VTA受体，以研究受体功能随时间的变化11。CIS-FSCV也可以被修改为测量5-HT和去甲肾上腺素（NE）24，25。CIS-FSCV也非常适合清醒，行为实验，并且可以与光遗传学方法26，27相结合。重要的是要注意，电诱发的释放事件与在自由移动研究中经常观察到的瞬态释放事件不同，在麻醉准备中较少见。例如，瞬态释放事件可能不一定由多巴胺神经元的直接去极化驱动，这与电诱发的释放事件不同28。因此，相位神经活动可能与通过FSCV检测到的多巴胺释放事件分离。此外，光学诱发的DA释放已被证明与电诱发的DA释放事件不同。最近对光学和电诱发刺激的比较表明，电诱发刺激产生相位DA释放的多突触调节，而光学诱发刺激可以将刺激限制在更具体的电路29。
最近的一些方法已经采用光遗传学和荧光方法来研究体内快速多巴胺动力学的电路30。例如，Sun及其同事最近的研究表明，黑质中多巴胺神经元的光遗传学刺激在纹状体中产生DA的快速升高，如通过表达G蛋白偶联受体激活的基于DA（GRABDA）传感器30。光遗传学和荧光方法的组合可用于刺激或抑制VTA的特定传入输入，同时测量NAc中的DA释放。CIS-FSCV不能像光遗传学刺激那样特异性地刺激传入物，但它具有优势，因为它可以解决VTA内有关突触前和突触后受体的问题。虽然荧光和FSCV方法都具有足够的时间分辨率（亚秒）和对DA的敏感性（1-10 nM）来比较测量相位DA释放30，31的变化，但FSCV可能比体内相位DA的荧光监测具有一个优势，即不需要遗传操作来记录。事实上，CIS-FSCV实验可以在数小时内完成，而光遗传学和荧光方法的组合需要足够的时间（数周）才能使用病毒构建体进行充分的表达。
CIS-FSCV的一个关键好处是，可以在完整的大脑中研究相位DA释放的特异性VTA受体调节，这基于其他体内研究，这些研究要么测量VTA神经元的电生理特性，要么建立在体外研究的基础上，评估相位DA释放的突触前调节3，12。CIS-FSCV的一个警告是，这些记录必须在相对富含DA的地区进行。这有两个原因:首先，FSCV灵敏度有一些限制，它只能检测纳摩尔范围内和高于6，19的DA浓度。其次，FSCV难以将去甲肾上腺素与DA分离，因为它们的循环伏安图几乎相同。因此，这些研究可能仅限于评估具有高DA的区域，例如内侧前额叶皮层的某些部分，NAc，纹状体和嗅结节32。未来的研究可能能够采用一些先进的FSCV方法，可以更好地区分DA和NE，以及其他电活性神经递质，如腺苷33和血清素12。

相位多巴胺（DA）从腹侧被盖区（VTA）释放到伏隔核（NAc）在奖励相关行为中起着至关重要的作用。VTA DA神经元从强直性放电（3-8 Hz）切换到突发式放电（>14Hz）1，从而在NAc中产生相位DA释放。VTA表达多种躯体外胚层受体，这些受体处于有利地位，可以控制从强直到爆发发射的转变2，3，4，5。确定这些受体中的哪些以及它们各自的输入控制相位DA释放将加深我们对奖励相关电路如何组织的理解。这里描述的方法，结合输注和刺激与快速扫描循环伏安法（CIS-FSCV）的目的是快速而稳健地评估VTA受体在驱动相位DA释放方面的功能。
术语联合输注和刺激（CIS）是指在药理学上操纵一组神经元（此处为VTA）上的受体并刺激这些神经元以研究受体的功能。在麻醉的大鼠中，我们电刺激VTA以在NAc核心中唤起大相DA信号（1-2μM），如快速扫描循环伏安法（FSCV）测量的那样。在刺激部位输注药物（即受体激动剂/拮抗剂）可用于通过观察诱发的相位DA释放的后续变化来测量VTA受体的功能。FSCV是一种电化学方法，具有高空间（50-100μm）和时间（10 Hz）分辨率，非常适合测量与奖励相关的相位DA事件6，7。该分辨率比其他体内神经化学测量（如微透析）更精细。因此，CIS-FSCV一起非常适合评估VTA受体对相位多巴胺释放的调节。
研究VTA受体功能的一种常见方法是使用电生理学方法的组合来解决这些受体如何改变神经元的放电速率1，8。这些研究对于了解哪些受体参与驱动DA激活时放电非常有价值。然而，这些研究只能表明在轴突末端下游可能发生的情况（即神经递质的释放）。CIS-FSCV建立在这些电生理学研究的基础上，通过回答VTA爆裂放电，相位DA释放的输出如何受到位于VTA树突和细胞体上的受体的调节。因此，CIS-FSCV非常适合在这些电生理学研究的基础上进行。例如，烟碱受体激活可以在VTA9中诱导爆裂，并且使用麻醉大鼠中的CIS-FSCV来证明烟碱乙酰胆碱受体（nAChR）在VTA中的激活也控制NAc10，11中的相位DA释放。
相位DA调节的机制检查也通常使用切片制剂以及药物的浴液应用进行研究。这些研究通常集中在从多巴胺末端释放的相位DA的突触前调节上，因为细胞体通常从切片12中移除。这些制剂对于研究突触前受体对多巴胺末端的影响很有价值，而CIS-FSCV更适合研究躯体突触受体对多巴胺神经元的影响，以及VTA的突触前输入。这种区别很重要，因为VTA中的体外胚层受体激活可能与NAc突触前受体激活具有不同的效果。事实上，阻断NAc中的多巴胺能突触前nAChRs可以提高爆发13期间的相位多巴胺释放，而VTA somatodendritc nAChRs10，11则相反。
CIS-FSCV是研究VTA受体调节相位DA释放能力的理想方法。重要的是，这种方法可以在完整的大鼠中进行，无论是麻醉还是自由移动。这种方法适用于急性研究，研究基线状态10，14下的受体功能以及可以评估药物暴露或行为操作后受体功能变化的长期研究11，15。

<table><tbody><tr><td>&lt;strong&gt;Electrode Filling Solution/Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micropipette</td><td>World Precision Instruments</td><td>MF286-5 (28 gauge)</td><td></td></tr><tr><td>Potassium Acetate</td><td>Sigma</td><td>236497-100G</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P3911-25G</td><td></td></tr><tr><td>&lt;strong&gt;Electrode Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Carbon fiber</td><td>Thornel</td><td>T650</td><td></td></tr><tr><td>Electrode puller</td><td>Narishige International</td><td>PE-22</td><td>Note: horizontal pullers can be used as well</td></tr><tr><td>Glass capillary</td><td>A-M systems</td><td>626000</td><td></td></tr><tr><td>Insulated wires for electrodes</td><td>Weico Wire and Cable Incorporated</td><td>UL 1423</td><td>Length; 10 cm; diameter,0.4mm; must get custom made; insulated material should cover 5 cm of the wire</td></tr><tr><td>Light Microscope (for viewing and cutting electrode)</td><td>Fischer Scientific</td><td>M3700</td><td></td></tr><tr><td>Pin</td><td>Phoenix Enterprises</td><td>HWS1646</td><td>To be soldered onto the insuled electrode wire and reference electrode; connects to headstage</td></tr><tr><td>Putty</td><td>Alcolin</td><td>&lt;a target=&quot;_blank&quot; href=&quot;https://www.dickblick.com/items/23922-1003/&quot;&gt;23922-1003&lt;/a&gt;</td><td>Used to place electrode on while cutting the carbon fiber</td></tr><tr><td>Scalpal Blade</td><td>World Precision Instruments</td><td>500239</td><td>For cutting carbon fiber to the apprpriate length</td></tr><tr><td>Silver Wire</td><td>Sigma</td><td>327026-4G</td><td></td></tr><tr><td>&lt;strong&gt;FSCV Hardware/Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Faraday Cage</td><td>U-Line</td><td>H-3618 (36&quot; x 24&quot; x 42&quot;)</td><td></td></tr><tr><td>Potentiostat</td><td>Univ. of N. Carolina, Electronics Facility</td><td></td><td></td></tr><tr><td>Stimulating electrode</td><td>PlasticsOne</td><td>MS303/2-A/SPC</td><td>when ordering, request a 22 mm cut below pedestal</td></tr><tr><td>TarHeel HDCV Software</td><td>University of North Carolina-Chapel Hill</td><td>-</td><td>&lt;a href=&quot;https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/&quot; target=&quot;_blank&quot;&gt;https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/&lt;/a&gt; for ordering information</td></tr><tr><td>UEI breakout box</td><td>Univ. of N. Carolina, Electronics Facility</td><td></td><td>&lt;a href=&quot;https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/&quot; target=&quot;_blank&quot;&gt;https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/&lt;/a&gt; for ordering information</td></tr><tr><td>UEI power supply</td><td>Univ. of N. Carolina, Electronics Facility</td><td></td><td>&lt;a href=&quot;https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/&quot; target=&quot;_blank&quot;&gt;https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/&lt;/a&gt; for ordering information</td></tr><tr><td>&lt;strong&gt;Stimulator Hardware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Neurolog stimulus isolator</td><td>Digitimer Ltd.</td><td>DS4</td><td>Neurolog 800A</td></tr><tr><td>&lt;strong&gt;Infusion/Stimulation Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Infusion Pump</td><td>New Era Syringe Pump</td><td>NE-300</td><td></td></tr><tr><td>Internal Cannula</td><td>PlasticsOne</td><td>C315I/SPC INTERNAL 33GA</td><td></td></tr><tr><td>Microliter Syringe</td><td>Hamilton</td><td>80308</td><td></td></tr><tr><td>Tubing</td><td>PlasticsOne</td><td>C313CT/ PKG TUBING 023 X 050 PE50</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cannula Holder</td><td>Kopf Instruments</td><td>1776 P-1</td><td></td></tr><tr><td>Cotton Tip Applicators</td><td>Vitality Medical</td><td>806</td><td></td></tr><tr><td>Electrode Holder</td><td>Kopf Instruments</td><td>1770</td><td></td></tr><tr><td>Heating Pad</td><td>Kent Scientific</td><td>RT-0501</td><td></td></tr><tr><td>Povidone Iodine</td><td>Vitality Medical</td><td>29906-004</td><td></td></tr><tr><td>Screws</td><td>Stoelting</td><td>Bone Anchor Screws/Pkg.of 100</td><td>1.59 mm O.D., 3.2 mm long</td></tr><tr><td>Silver wire reference with AgCl</td><td>InVivo Metric</td><td>E255A</td><td></td></tr><tr><td>Square Gauze</td><td>Vitality Medical</td><td>441408</td><td></td></tr><tr><td>Stereotax</td><td>Kopf Instruments</td><td>Model 902 (Dual Arm Bar)</td><td></td></tr><tr><td>&lt;strong&gt;Histological Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Formulin</td><td>Sigma</td><td>1004960700</td><td></td></tr><tr><td>Power supply</td><td>BK Precision</td><td>9110</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>80497</td><td></td></tr><tr><td>Tungsten microelectrode</td><td>MicroProbes</td><td>WE30030.5A3</td><td></td></tr><tr><td>&lt;strong&gt;Drugs for infusions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>((2&lt;em&gt;R&lt;/em&gt;)-amino-5-phosphonovaleric acid</td><td>Sigma Aldrich</td><td>A5282</td><td></td></tr><tr><td>N-methyl-D-aspartate</td><td>Sigma Aldrich</td><td>M3262</td><td></td></tr><tr><td>Mecamylamine hydrochloride (M9020-5mg)</td><td>Sigma Aldrich</td><td>M9020</td><td></td></tr><tr><td>Scopolamine hydrobromide (S0929-1g)</td><td>Sigma Aldrich</td><td>S0929</td><td></td></tr></tbody></table>

combined infusion and stimulation with fast-scan cyclic voltammetry (cis-fscv) to assess ventral tegmental area receptor regulation of phasic dopamine

相位多巴胺（DA）从腹侧被盖区（VTA）释放到伏隔核在奖励处理和强化学习中起着关键作用。了解VTA控制相位DA释放中的各种神经元输入如何能够更好地反映控制奖励处理和强化学习的电路。在这里，我们描述了一种将药物激动剂和拮抗剂的VTA插管内输注与刺激诱发的相位DA释放（联合输注和刺激，或CIS）相结合的方法，该方法通过体内快速扫描循环伏安法（FSCV）测量。在麻醉大鼠中使用CIS-FSCV，可以通过用装有套管的双极电极电刺激VTA来引起相位DA反应，同时记录在伏隔核核心中。药理激动剂或拮抗剂可以直接输注在刺激部位，以研究特异性VTA受体在驱动相位DA释放中的作用。CIS-FSCV的一个主要好处是VTA受体功能可以在体外研究的基础上在体内进行研究。

CIS-FSCV用于研究VTA N-甲基-D-天冬氨酸受体（NMDAR），烟碱乙酰胆碱受体（nAChRs）和毒蕈碱乙酰胆碱受体（mAChRs）在驱动NAc核心中相位DA释放中的功能。 图2 显示了阴性对照的代表性数据，输注0.9%盐水，在输注前（基线）和输注后9分钟（生理盐水）。 图2 显示了一个颜色图，y轴上有电位，x轴上有时间，z轴上有电流（表示为假色），电流与时间轨迹（IvT），以及在峰值诱发电应时拍摄的循环伏安图，以证明测量的分析物对应于DA。正如预期的那样，盐水输注不会改变受刺激的相位DA释放。
为了证明CIS-FSCV在使用激动剂和拮抗剂时可以产生双向效应，我们比较了NMDAR激动剂NMDA（500ng;图3A）到NMDAR拮抗剂AP5（1μg;图 3B）。NMDA输注产生受刺激相位DA释放的强劲增加（图3A，输注后9分钟），而NMDAR竞争性拮抗剂AP5（1μg）产生强劲的减少（图3B，输注后9分钟）。为了证明CIS-FSCV使用靶向不同类别乙酰胆碱受体的拮抗剂的效用，我们比较了输注非选择性，非竞争性nAChR拮抗剂甲黄酰胺（3μg;图4A）和非选择性，竞争性mAChR拮抗剂东莨菪碱（67μg;图 4B）。两种药物都产生了刺激的相位DA释放的强劲减少（图4，输注后9分钟）。图2、图3和图4的结果摘要在图5中重新绘制，其中基线周期是五次刺激的平均值，药物周期显示为基线平均值的百分比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60886/60886fig1.jpg"> 图1:VTA微输注前后记录的时间线。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60886/60886fig1large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60886/60886fig2.jpg"> 图2:单只雄性Sprague Dawley大鼠在NAc核心中受刺激的相位DA释放的基线（左）和盐水（载体）输注（右）的代表性颜色和IvT图。 蓝色条代表刺激。基线记录发生在t = 0时，在将内部套管放置在双极刺激器的引导套管中之前。盐水记录在输注后9分钟（t = 9）进行。循环伏安图插图对应于IvT图的峰值，显示0.6 V时的氧化峰和-0.2 V时的峰还原，表明DA。输注生理盐水 VTA 后，不应观察到刺激诱发释放的变化。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60886/60886fig2large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60886/60886fig3.jpg"> 图3:输注NMDAR激动剂（NMDA）和拮抗剂（AP5）的影响。（A）在单个雄性Sprague Dawley大鼠的NAc核心中受刺激的相位DA释放的基线（左）和500ng NMDAR激动剂输注（右）的代表性颜色和IvT图。NMDA输注增加了刺激的相位DA释放（输注后9分钟记录）。（B） 在单个雄性 Sprague Dawley 大鼠中 NAc 核心中受刺激的相位 DA 释放时，基线（左）和 1 μg NMDAR 拮抗剂 （2R）-氨基-5-膦源戊酸 （AP5） 输注（右）的代表性颜色和 IvT 图。AP5输注减少了刺激的相位DA释放（输注后9分钟记录）。基线记录发生在t = 0时，在将内部套管放置在双极刺激器的引导套管中之前。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60886/60886fig3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60886/60886fig4.jpg"> 图4:输注甲菀胺和东莨菪碱的影响。 （A） 在单个雄性 Sprague Dawley 大鼠的 NAc 核心中刺激相位 DA 释放时，基线（左）和 3 μg 非选择性烟碱乙酰胆碱受体拮抗剂甲壬胺 （MEC） 输注（右）的代表性颜色和 IvT 图。（B） 在单个雄性 Sprague Dawley 大鼠的 NAc 核心中受刺激相位 DA 释放时，基线（左）和非选择性毒蕈碱乙酰胆碱受体拮抗剂东莨菪碱 （SCOP） 输注（右）的基线（左）和 67 μg 的代表性颜色和 IvT 图。基线记录发生在t = 0时，在将内部套管放置在双极刺激器的引导套管中之前。MEC和SCOP记录在输注后9分钟进行。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60886/60886fig4large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60886/60886fig5.jpg"> 图5:显示药物效应随时间变化的数据摘要。 输注前期（基线）在五次刺激中平均，输注后期（从t = 3开始）表示为基线的百分比。在输注后9 min，我们观察到诱发的DA信号在盐水输注后为基线的103%，NMDA输注后为196%，AP5输注后为18%，MEC输注后为49%，SCOP输注后为43%。n = 每个条件 1。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60886/60886fig5large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine at JoVE.c

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

该协议的目标是直接操纵腹侧被盖区域受体，以研究它们对亚秒多巴胺释放的贡献。

联合输注和刺激与快速扫描循环伏安法（CIS-FSCV）评估腹侧被盖区域受体对相位多巴胺的调节

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...

combined-infusion-stimulation-with-fast-scan-cyclic-voltammetry-cis

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

3.0K 次观看.  Elizabethtown College. 该协议的目标是直接操纵腹侧被盖区域受体，以研究它们对亚秒多巴胺释放的贡献。

视频: Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET:  Dopamine Movies of Cigarette Smoking

We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8 that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7 to a conventional model 9. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.

We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking.

创建动态图像的短命多巴胺波动与LP-ntPET：多巴胺电影吸烟

We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute ...

科研

行为学

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique with high spatial and temporal (sub-second) resolution that has been utilized to examine phasic DA release in several types of preparations. In vitro experiments in single-cells and brain slices and in vivo experiments in anesthetized rodents have been used to identify mechanisms that mediate dopamine release and uptake under normal conditions and in disease models. Over the last 20 years, in vivo FSCV experiments in awake, freely moving rodents have also provided insight of dopaminergic mechanisms in reward processing and reward learning. One major advantage of the awake, freely moving preparation is the ability to examine rapid DA fluctuations that are time-locked to specific behavioral events or to reward or cue presentation. However, one limitation of combined behavior and voltammetry experiments is the difficulty of dissociating DA effects that are specific to primary rewarding or aversive stimuli from co-occurring DA fluctuations that mediate reward-directed or other motor behaviors. Here, we describe a combined method using in vivo FSCV and intra-oral infusion in an awake rat to directly investigate DA responses to oral tastants. In these experiments, oral tastants are infused directly to the palate of the rat &#8211; bypassing reward-directed behavior and voluntary drinking behavior &#8211; allowing for direct examination of DA responses to tastant stimuli.

Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

考试多巴胺快速与动态快速扫描循环伏安法在口内呈味物质管理清醒大鼠

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

突触前多巴胺纹状体脑片的快速扫描循环伏安动态

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

神经科学

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

单细胞的多巴胺释放，同时电压钳和电流分析法测量

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

多巴胺的释放突触前货箱码头在个别的可视化与FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

生物学

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol for a real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the so-called neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easy-to-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

在小鼠的文室内使用光遗传学的两种不同的实时位置偏好范式

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...

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 ...

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

使用QNsim1.0从电刺激多巴胺神经传递数据建模快速扫描循环伏安数据

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...