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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T., 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=Hitchhiker's+Guide+to+Voltammetry:+Acute+and+Chronic+Electrodes+for+in+vivo+Fast-Scan+Cyclic+Voltammetry.">Hitchhiker's Guide to Voltammetry: Acute and Chronic Electrodes for in vivo Fast-Scan Cyclic Voltammetry.</a> ACS Chemical Neuroscience. 8 (2), 221-234 (2017).</li><li>Sabeti, J., Gerhardt, G. A., Zahniser, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloral+hydrate+and+ethanol,+but+not+urethane,+alter+the+clearance+of+exogenous+dopamine+recorded+by+chronoamperometry+in+striatum+of+unrestrained+rats.">Chloral hydrate and ethanol, but not urethane, alter the clearance of exogenous dopamine recorded by chronoamperometry in striatum of unrestrained rats.</a> Neuroscience Letters. 343 (1), 9-12 (2003).</li><li>Masuzawa, 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=Pentobarbital+inhibits+ketamine-induced+dopamine+release+in+the+rat+nucleus+accumbens:+a+microdialysis+study.">Pentobarbital inhibits ketamine-induced dopamine release in the rat nucleus accumbens: a microdialysis study.</a> Anesthesia &amp; Analgesia. 96 (1), 148-152 (2003).</li><li>Montague, P. 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=Dynamic+gain+control+of+dopamine+delivery+in+freely+moving+animals.">Dynamic gain control of dopamine delivery in freely moving animals.</a> Journal of Neuroscience. 24 (7), 1754-1759 (2004).</li><li>Keithley, R. B., 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=Higher+sensitivity+dopamine+measurements+with+faster-scan+cyclic+voltammetry.">Higher sensitivity dopamine measurements with faster-scan cyclic voltammetry.</a> Analytical Chemistry. 83 (9), 3563-3571 (2011).</li><li>Jackson, B. P., Dietz, S. 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=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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electrode Filling Solution/Supplies</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>Electrode Supplies</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><a target="_blank" href="https://www.dickblick.com/items/23922-1003/">23922-1003</a></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>FSCV Hardware/Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>U-Line</td>
			<td>H-3618 (36&#34; x 24&#34; x 42&#34;)</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><a href="https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/" target="_blank">https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/</a> for ordering information</td>
		</tr>
		<tr>
			<td>UEI breakout box</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td><a href="https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/" target="_blank">https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/</a> for ordering information</td>
		</tr>
		<tr>
			<td>UEI power supply</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td><a href="https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/" target="_blank">https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/</a> for ordering information</td>
		</tr>
		<tr>
			<td>Stimulator Hardware</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>Infusion/Stimulation Supplies</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>Surgical Supplies</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>Histological Supplies</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>Drugs for infusions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>((2R)-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?...

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

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

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

科研

神经科学

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS:使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

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 ...

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

偏置电压，循环伏安，神经接口电阻抗光谱

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive ...

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 ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

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 ...