Name: making, testing, and using potassium ion selective microelectrodes in tissue slices of adult brain
Uploaded: 2018-05-07T14:45:00.000+00:00
Duration: 11 min 20 s
Description: Watch this Scientific Journal Video about Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain at JoVE.com

Khakh 实验室得到了 NIH MH104069 的支持。这个实验室是由 NIH NS030549 支持的。J.C.O. 感谢 NIH T32 神经集成电路训练补助金 (NS058280)。

所有动物实验都是按照美国国家卫生研究院的实验室动物护理和使用指南进行的, 并得到了加州大学洛杉矶分校校长动物研究委员会的批准。所有的老鼠都是在12小时的昏暗环境中使用食物和水的ad 随意。所有动物都是健康的, 没有明显的行为变化, 没有参与以前的研究, 并在光周期中牺牲。实验数据从成年小鼠 (6-8 周大的所有实验) 中采集。
1. K+选择性电极的制备
<ol><li>硼硅酸盐玻璃的硅烷化<ol>
<li>从包装中取出足够的玻璃毛细血管, 放入50毫升锥形管。用1米 hcl 填充锥形管顶部. 在 hcl 中用温和的搅拌方法清洗电极一夜之间或至少6小时。</li>
		<li>用70% 乙醇简单冲洗毛细血管, 然后在100-120 摄氏度内完全干燥6-8 小时。在使用无水硫酸钙干燥剂的容器中贮存清洗过的毛细血管长达4周。</li>
		<li>在硅烷化之前, 用微电极拉拔毛细血管到细尖端。我们使用的电极直径约为2-5 微米。总是用手套处理冲洗过的毛细血管, 因为皮肤上的油脂会干扰硅烷化。</li>
		<li>将电极放入玻璃容器中, 使电极从底部升高以防止尖端破损。使用耐高压胶带或类似胶带将电极固定在容器上。</li>
		<li>使用氮气替换方法从容器中移除大约0.5 毫升 5% dichlorodimethylsilane (DDS) 硅烷化溶液 (请参见图 2)。用氮气填充气球, 然后将注射器、管子和针头连接到气球上。将该针插入 dds 容器, 同时通过较长的针将 DDS 绘制到单独的注射器中。</li>
		<li>将硅烷化溶液滴在吸管的尖端, 立即盖上。将容器持有电极与硅烷化溶液放入预热 (170-180 °c) 实验室烤箱10-12 小时或200-220 摄氏度30分钟</li>
		<li>孵化后, 关闭烤箱, 然后从烤箱中取出盘子。从孵化器中取出盘子时要小心, 因为它非常热。将盘子放在室温下的长凳上10-15 分钟, 让玻璃器皿冷却。</li>
		<li>从盘子中取出电极 (使用剃刀刀片或手术刀刀片来剪切胶带), 并将其放入干燥剂填充的密闭容器中。硅烷化电极保持水分可用于1周以下硅烷化。</li>
	</ol>
</li>
	<li>启动电极<ol>
<li>准备 K+载体鸡尾酒的库存解决方案: 5% 瓦特/v valinomycin, 93% 伏/v 12-二甲基3硝基苯, 2% 瓦特/v 钾四 (4-氯苯基) 硼酸盐11。这个解决方案是一个淡黄色的颜色。在室温下保持密闭、不透明的容器。如果正确存储此解决方案可以持续许多个月。</li>
		<li>准备一个10毫米 HEPES 缓冲300毫米氯化钠在 pH 值7.4 的股票溶液。使用与注射器连接的28G 丙纶尖端, 将电极固定在夹持器和回填用缓冲氯化钠。观察生理盐水溶液已达到微电极尖端的末端。确认微电极没有大气泡, 可能干扰电流的流动。</li>
		<li>用手术刀或剃刀刀片的钝面将电极的尖端折断到大约10-20 µm 宽。</li>
		<li>使用微, 在微电极尖端附近应用 K+载体的小水滴 (~ 0.1 µl)。如果电极已正确硅烷化液滴将被吸收到破碎的尖端。用 K+载体将电极填充到1-2 毫米, 并使用纸巾去除多余的部分。</li>
	</ol>
</li>
</ol>2. K+选择性电极的校准
<ol><li>校准解决方案的准备<ol>
<li>用氯化钾代替氯化钠, 制备等渗透人工脑脊液中不同浓度氯化钾的溶液 (ACSF)。我们使用了0.1、1、4.5、10和100毫米 K+ ACSF, 用于校准电极。这些校准解决方案的食谱列在表 1中。</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="2">化学</td>
			<td>M w</td>
			<td rowspan="2">最后 mM</td>
			<td rowspan="2">0.1 毫米 [K +]</td>
			<td rowspan="2">1毫米 [K +]</td>
			<td rowspan="2">4.5 毫米 [K +]</td>
			<td rowspan="2">10毫米 [K +]</td>
			<td rowspan="2">100毫米 [K +]</td>
		</tr>
<tr>
<td>(克/摩尔)</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>58.44</td>
			<td>不同</td>
			<td>1.51 克</td>
			<td>1.50 克</td>
			<td>1.44 克</td>
			<td>1.4 克</td>
			<td>0.345 克</td>
		</tr>
<tr>
<td>氯化钾</td>
			<td>1米库存</td>
			<td>不同</td>
			<td>20µl</td>
			<td>200µl</td>
			<td>900µl</td>
			<td>2毫升</td>
			<td>20毫升</td>
		</tr>
<tr>
<td>CaCl2
</td>
			<td>1米库存</td>
			<td>2</td>
			<td colspan="5">400µl</td>
		</tr>
<tr>
<td>氯化镁2
</td>
			<td>1米库存</td>
			<td>1</td>
			<td colspan="5">200µl</td>
		</tr>
<tr>
<td>NaH2PO4
</td>
			<td>119.98</td>
			<td>1。2</td>
			<td colspan="5">0.29 克</td>
		</tr>
<tr>
<td>NaHCO3
</td>
			<td>84.01</td>
			<td>26</td>
			<td colspan="5">0.437 克</td>
		</tr>
<tr>
<td>d-葡萄糖</td>
			<td>180.16</td>
			<td>10</td>
			<td colspan="5">0.360 克</td>
		</tr>
<tr>
<td>水</td>
			<td colspan="7">q.s. 200 毫升</td>
		</tr>
</tbody></table>表1。钾校准解决方案
<ol start="2"><li>微电极校准<ol>
<li>在开始实验前至少20分钟冒泡所有解决方案, 95% O2/5% CO2 。以每分钟3毫升的速率开始灌注4.5 毫米 [K+] ACSF 的浴缸。将 K+选择性电极放入附着在机械手上电极 headstage 的电极支架上。此 headstage 连接到适当的放大器。将电极尖端插入浴缸灌流液。</li>
		<li>确保银/AgCl 接地电极沐浴在相同的溶液中, 并且流量是稳定的。以逐步的方式应用校准解决方案, 并记录在 mV 中的电位变化在电极尖端。在切换到下一个解决方案之前, 等待电极尖端的电位达到稳定值</li>
		<li>测量稳态电压的变化, 以响应对电极尖端的校准解决方案的应用。确认在 [K+] 中, 电极响应的斜率至少为52且不大于 58 mV。</li>
	</ol>
</li>
</ol>3. 急性海马脑切片的制备
<ol><li>切片溶液的制备<ol>
<li>准备500毫升蔗糖切割液, 由: 194 mM 蔗糖组成, 30 毫米氯化钠, 4.5 毫米氯化钾, 10 毫米 d-葡萄糖, 1 毫米氯化镁2, 1.2 毫米 NaH2PO4和26毫米 NaHCO3, 290-300 mOsm, 饱和与 95% O2和 5% CO2。</li>
		<li>准备1-2 公升记录解决方案 (ACSF), 包括: 124 毫米氯化钠, 4.5 毫米氯化钾, 1 毫米氯化镁2, 10 毫米 d-葡萄糖, 2 毫米 CaCl2, 1.2 毫米 NaH2PO4, 26 毫米 NaHCO3;pH 值 7.3-7.4 (冒泡后), 290-300 mOsm, 饱和与 95% O2和 5% CO2。用录音溶液填充含有脑切片夹室的烧杯, 并保持在32-34 摄氏度。用冰水来填满 vibratome 室。</li>
	</ol>
</li>
	<li>急性切片制备<ol>
<li>深麻醉一只老鼠, 把它放在钟罐子 precharged 与2-3 毫升异氟醚。检查脚趾捏反射, 如果不响应, 迅速斩首它使用一对锋利的剪刀或断头台, 因为你的动物协议要求。</li>
		<li>用剪刀从头骨的尾部部分做一个2-3 厘米的切口, 沿着中线切开头皮。当手动缩回头皮, 使两个1厘米水平切口从孔大口径沿头骨两侧。然后, 使用细剪, 切开切口长度的头骨, 沿中线从头骨后面的鼻子。</li>
		<li>使用在中线附近插入的细钳, 收回被切开的头骨在两个部分。从头骨中提取小鼠大脑, 用刀片去除小脑和嗅球, 分别位于脑尾部和延髓部分。这些都可以通过大的裂隙来识别, 它们与皮层分离。</li>
		<li>使用超级胶水将脑块安装到 vibratome 托盘上。用冰冷切削液填充 vibratome 托盘。</li>
		<li>在冠状面上切开组织切片, 厚度为300µm。通常可以收集6个冠状海马切片。</li>
		<li>每节切完后, 立即将切片转移到片托烧杯上, 32-34°C。在取下包含该节的烧杯之前, 将这些节保持在20分钟的温度, 在录制前至少20-30 分钟将其放置在室温下。</li>
	</ol>
</li>
</ol>4. 电诱发 K+动力学的测量
<ol><li>设置切片准备<ol>
<li>用巴斯德吸管轻轻地将大脑切片放在浴缸中, 用带有尼龙弦的铂竖琴轻轻地握住它。</li>
		<li>确保双极刺激电极的尖端彼此平行, 并与切片的平面保持水平。慢慢地, 在6-7 秒的过程中, 将电极插入 CA3 层 radiatum 大约40-50 µm 深刺激谢弗络。在冠状部分, CA3 可以近似地被辨认作为海马的部分正常侧向颗粒细胞层在海马膝, 与地层 radiatum 下落的内侧和腹侧到锥体细胞层数。</li>
		<li>小心地将 K+选择性电极插入 CA1 地层 radiatum 约50µm 深, 通过慢慢降低电极约3-4 秒。在对切片施加刺激之前, 允许电位稳定在电极上: 这通常需要5到10分钟。如果切片在胞外 K+中呈现自发的变化, 则用新切片丢弃并重复此过程。</li>
	</ol>
</li>
	<li>测量诱发 K+版本<ol>
<li>应用电刺激 (8 脉冲) 的列车, 通过在数字记录响应时手动压制刺激器上的触发器。应用刺激在10赫兹和1毫秒脉宽, 从10µA 刺激幅度开始。</li>
		<li>在检测到最大 K+响应振幅的情况下, 将增加的刺激振幅提高2倍。如果看不到任何响应, 请将 K+电极的位置以100µm 的增量更接近刺激站点。</li>
		<li>确定产生半最大响应的刺激振幅。根据我们的经验, 这是在40-160 µA 之间, 取决于准备质量, 动物的年龄, 和刺激电极和 K+选择性电极之间的距离。</li>
		<li>在同一切片中, 使用一个比产生半最大响应的振幅低一步的刺激振幅 (例如, 如果80µA 产生一个半最大响应, 使用40µA) 应用刺激列车的脉冲数量增加。最初, 我们使用了 1, 2, 4, 8, 16, 32, 64 和128脉冲的火车。</li>
		<li>要确认 K+信号是由电刺激的谢弗络浴的动作电位发射介导的, 请在 ACSF 中应用0.5 µM TTX 10 分钟, 并重复刺激协议。不应观察诱发反应。</li>
		<li>在完成切片实验后, 确认电极在校准解决方案中重新校准电极以保证其响应, 并确保响应在初始校准时不偏离10% 以上。</li>
	</ol>
</li>
</ol>

<ol>
	<li>McDonough, A. A., Youn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+homeostasis:+The+knowns,+the+unknowns,+and+the+health+benefits.">Potassium homeostasis: The knowns, the unknowns, and the health benefits.</a> Physiol Bethesda Md. 32 (2), 100-111 (2017).</li><li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion channels of excitable membranes. , 507 (2001).</li><li>Kofuji, P., Ceelen, P., Zahs, K. R., Surbeck, L. W., Lester, H. A., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+inactivation+of+an+inwardly+rectifying+potassium+channel+(Kir4.1+subunit)+in+mice:+Phenotypic+impact+in+retina.">Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: Phenotypic impact in retina.</a> J Neurosci. 20 (15), 5733-5740 (2000).</li><li>Sibille, J., Dao Duc, K., Holcman, D., Rouach, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroglial+potassium+cycle+during+neurotransmission:+role+of+Kir4.1+channels.">The neuroglial potassium cycle during neurotransmission: role of Kir4.1 channels.</a> PLoS Comput Biol. 11 (3), e1004137 (2015).</li><li>Tong, X., 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=Astrocyte+Kir4.1+ion+channel+deficits+contribute+to+neuronal+dysfunction+in+Huntington's+disease+model+mice.">Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.</a> Nat Neurosci. 17 (5), 694-703 (2014).</li><li>Datta, D., Sarkar, K., Mukherjee, S., Meshik, X., Stroscio, M. A., Dutta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene+oxide+and+DNA+aptamer+based+sub-nanomolar+potassium+detecting+optical+nanosensor.">Graphene oxide and DNA aptamer based sub-nanomolar potassium detecting optical nanosensor.</a> Nanotechnology. 28 (32), 325502 (2017).</li><li>Bandara, H. M. 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=Palladium-Mediated+Synthesis+of+a+Near-Infrared+Fluorescent+K++Sensor.">Palladium-Mediated Synthesis of a Near-Infrared Fluorescent K+ Sensor.</a> J Org Chem. 82 (15), 8199-8205 (2017).</li><li>Depauw, 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=A+highly+selective+potassium+sensor+for+the+detection+of+potassium+in+living+tissues.">A highly selective potassium sensor for the detection of potassium in living tissues.</a> Chem Weinh Bergstr Ger. 22 (42), 14902-14911 (2016).</li><li>Machado, 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=Biofouling-Resistant+Impedimetric+Sensor+for+Array+High-Resolution+Extracellular+Potassium+Monitoring+in+the+Brain.">Biofouling-Resistant Impedimetric Sensor for Array High-Resolution Extracellular Potassium Monitoring in the Brain.</a> Biosensors. 6 (4), (2016).</li><li>Rose, M. C., Henkens, 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=Stability+of+sodium+and+potassium+complexes+of+valinomycin.">Stability of sodium and potassium complexes of valinomycin.</a> Biochim Biophys Acta BBA - Gen Subj. 372 (2), 426-435 (1974).</li><li>Ammann, D., Chao, P., Simon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valinomycin-based+K++selective+microelectrodes+with+low+electrical+membrane+resistance.">Valinomycin-based K+ selective microelectrodes with low electrical membrane resistance.</a> Neurosci Lett. 74 (2), 221-226 (1987).</li><li>Amzica, F., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+glial+membrane+potentials+during+sleep+and+paroxysmal+oscillations+in+the+neocortex.">Neuronal and glial membrane potentials during sleep and paroxysmal oscillations in the neocortex.</a> J Neurosci. 20 (17), 6648-6665 (2000).</li><li>Amzica, F., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+significance+of+K-complexes.">The functional significance of K-complexes.</a> Sleep Med Rev. 6 (2), 139-149 (2002).</li><li>MacVicar, B. A., Feighan, D., Brown, A., Ransom, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+optical+signals+in+the+rat+optic+nerve:+role+for+K(+)+uptake+via+NKCC1+and+swelling+of+astrocytes.">Intrinsic optical signals in the rat optic nerve: role for K(+) uptake via NKCC1 and swelling of astrocytes.</a> Glia. 37 (2), 114-123 (2002).</li><li>Chever, O., Djukic, B., McCarthy, K. D., Amzica, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+Kir4.1+channel+in+excess+potassium+clearance:+an+in+vivo+study+on+anesthetized+glial-conditional+Kir4.1+knock-out+mice.">Implication of Kir4.1 channel in excess potassium clearance: an in vivo study on anesthetized glial-conditional Kir4.1 knock-out mice.</a> J Neurosci. 30 (47), 15769-15777 (2010).</li><li>Hall, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-selective+membrane+electrodes:+A+general+limiting+treatment+of+interference+effects.">Ion-selective membrane electrodes: A general limiting treatment of interference effects.</a> J Phys Chem. 100 (17), 7230-7236 (1996).</li><li>Haack, N., Durry, S., Kafitz, K. W., Chesler, M., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-barreled+and+Concentric+Microelectrodes+for+Measurement+of+Extracellular+Ion+Signals+in+Brain+Tissue.">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.</a> J Vis Exp. (103), e53058 (2015).</li><li>Larsen, B. R., MacAulay, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kir4.1-mediated+spatial+buffering+of+K(+):+Experimental+challenges+in+determination+of+its+temporal+and+quantitative+contribution+to+K(+)+clearance+in+the+brain.">Kir4.1-mediated spatial buffering of K(+): Experimental challenges in determination of its temporal and quantitative contribution to K(+) clearance in the brain.</a> Channels Austin Tex. 8 (6), 544-550 (2014).</li><li>Mei, 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=Long-term+in+vivo+recording+of+circadian+rhythms+in+brains+of+freely+moving+mice.">Long-term in vivo recording of circadian rhythms in brains of freely moving mice.</a> Proceedings of the National Academy of Sciences. 115, 4276-4281 (2018).</li></ol>

作者没有什么可透露的。

我们在这里描述的方法使我们能够评估 K+动力学, 以响应电刺激的谢弗络在急性海马切片的成年小鼠。我们准备 K+离子选择性电极的方法类似于前面描述的过程12,13,14,15。然而, 这种方法优于替代电极配置, 因为它是快速和复杂的准备 K+选择性电极。在适当的校准后, 发现这些电...

钾离子浓度在大脑中受到严格的调节, 它们的波动对所有细胞的静止膜电位有很大的影响。根据这些重要的贡献, 生物学的一个重要目标是确定细胞和生物物理机制, 用于在身体的不同器官的细胞外空间中严格调节 K+的浓度1,2. 这些研究的一个重要要求是能够准确地测量 K+浓度。虽然在健康和患病状态下, 许多有助于大脑中钾稳态的成分已经被确认为3,45, 由于专业性质的原因, 进一步的进展已经减慢。制备离子选择性电极钾的方法。微电极传感器表示用于测量 K+浓度体外、组织切片和体内中的金标准。
用于 k+监视的更新方法正在使用光学传感器进行开发, 但这些检测不到与生物学相关的 k+浓度范围, 也未在生物系统中进行全面审查, 尽管初步结果显示有希望的6,7,8。与光学传感器相比, 电极从根本上被限制为离子的点源测量, 虽然电极阵列可以改善空间分辨率9。本文重点介绍了用于监测 K+动力学的单桶微电极传感器。
在这项工作中, 我们报告详细的逐步过程, 使 K+选择性电极, 使用基于 valinomycin 的钾载体, 允许高度选择性 (104折叠 k+到 Na+选择性) k+在膜上运动10。valinomycin 是一种自然发生的多肽, 它充当 k+渗透性孔隙, 并使 k+的流量降低到其电化学梯度。我们还描述了如何校准电极, 如何存储和使用它们, 最后如何部署它们来测量成年小鼠海马脑切片中的 K+浓度动态。使用此类电极和基因修饰小鼠缺乏特定的离子通道来调节胞外 K+动力学应该揭示神经系统使用的细胞机制来控制 k 的环境浓度。在胞外环境中的+ 。

<table><tbody><tr><td>Vibratome</td><td>DSK</td><td>Microslicer Zero 1</td><td></td></tr><tr><td>Mouse: C57BL/6NTac inbred mice</td><td>Taconic</td><td>Stock#B6</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX51</td><td></td></tr><tr><td>Electrode puller</td><td>Sutter</td><td>P-97</td><td></td></tr><tr><td>Ag/AgCl ground pellet</td><td>WPI</td><td>EP2</td><td></td></tr><tr><td>pCLAMP10.3</td><td>Molecular Devices</td><td>n/a</td><td></td></tr><tr><td>Custom microfil 28G tip</td><td>World precision instruments</td><td>CMF28G</td><td></td></tr><tr><td>Tungsten Rod</td><td>A-M Systems</td><td>716000</td><td></td></tr><tr><td>Bipolar stimulating electrodes</td><td>FHC</td><td>MX21XEW(T01)</td><td></td></tr><tr><td>Stimulus isolator</td><td>World precision instruments</td><td>A365</td><td></td></tr><tr><td>Grass S88 Stimulator</td><td>Grass Instruments Company</td><td>S88</td><td></td></tr><tr><td>Borosilicate glass pipettes</td><td>World precision instruments</td><td>1B150-4</td><td></td></tr><tr><td>A to D board</td><td>Digidata 1322A</td><td>Axon Instruments</td><td></td></tr><tr><td>Signal Amplifier</td><td>Multiclamp 700A or 700B</td><td>Axon Instruments</td><td></td></tr><tr><td>Headstage</td><td>CV-7B Cat 1</td><td>Axon Instruments</td><td></td></tr><tr><td>Patch computer</td><td>Dell</td><td>n/a</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma</td><td>S0751</td><td></td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>21108</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>valinomycin</td><td>Sigma</td><td>V0627-10mg</td><td></td></tr><tr><td>1,2-dimethyl-3-nitrobenzene</td><td>Sigma</td><td>40870-25ml</td><td></td></tr><tr><td>Potassium tetrakis (4-chlorophenyl)borate</td><td>Sigma</td><td>60591-100mg</td><td></td></tr><tr><td>5% dimethyldichlorosilane in heptane</td><td>Sigma</td><td>85126-5ml</td><td></td></tr><tr><td>TTX</td><td>Cayman Chemical Company</td><td>14964</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma</td><td>H1758-500mL</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378-5kg</td><td></td></tr><tr><td>Pipette Micromanipulator</td><td>Sutter</td><td>MP-285 / ROE-200 / MPC-200</td><td></td></tr><tr><td>Objective lens</td><td>Olympus</td><td>PlanAPO 10xW</td><td></td></tr></tbody></table>

making, testing, and using potassium ion selective microelectrodes in tissue slices of adult brain

钾离子对细胞的静止膜电位有显著的促进作用, 因此胞外 K+浓度是细胞兴奋性的重要调节因子。细胞外 K+的浓度改变会影响静止膜电位和细胞兴奋性, 方法是将闭合的、开放的和灭活状态之间的平衡转移到有作用电位的电压依赖离子通道上。启动和传导。因此, 直接测量健康和患病状态下的胞外 K+动态是很有价值的。在这里, 我们描述如何制作, 校准和使用单 K+选择性电极。我们将它们部署在成人海马脑切片中, 以测量电诱发 K+浓度动态。明智地使用此类电极是评估细胞和生物物理机制, 以控制神经系统细胞外 K+浓度所需的工具套件的重要组成部分。

对于细胞外 K+的选择性测量, 我们通过硅烷化清洁硼硅酸盐玻璃吸管(图 1A), 制备了具有疏水性层的离子选择性电极。此涂层使包含 valinomycin 的 k+载体在电极尖端上休息, 并且仅允许 k+通量通过在电极尖端的窄开口 (图 1B)。在用回填盐水溶液和 K+载体启动电极后, 可以对电极进行测试, 使其快速、?...

Watch this Scientific Journal Video about Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain at JoVE.com

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

成人脑组织切片中钾离子选择性电极的制备、检测及应用

钾离子有助于细胞的静止膜电位, 胞外 K+浓度是细胞兴奋性的重要调节因子。我们描述如何制作, 校准和使用单 K+选择性电极。使用此类电极可以测量成年海马切片中电诱发的 K+浓度动态。

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

making-testing-using-potassium-ion-selective-microelectrodes-tissue

Research

JoVE Journal

Neuroscience

12.0K 次观看.  University of California Los Angeles. 钾离子有助于细胞的静止膜电位, 胞外 K+浓度是细胞兴奋性的重要调节因子。我们描述如何制作, 校准和使用单 K+选择性电极。使用此类电极可以测量成年海马切片中电诱发的 K+浓度动态。

视频: Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

科研

神经科学

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

在使用单细胞RT - PCR技术的黑质神经元电压门控钾通道mRNA表达分析

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

医学

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

切片热门:成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

脑组织中的双管和同心微电极用于测量细胞外离子的信号

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

微胶质过程对急性脑片 atp 或血清素的双光子成像

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Characterizing Extracellular Space in a Brain Slice Using Ion-Selective and Iontophoresis Microelectrodes

Source:&#160; Odackal, J., et al. <a href="https://app.jove.com/v/55755">Real-time iontophoresis with tetramethylammonium to quantify volume fraction and tortuosity of brain extracellular space.</a> J. Vis. Exp. (2017)
This video demonstrates how to characterize the extracellular space in a brain slice by using ion-selective and iontophoresis microelectrodes to measure and compare electrical signals. This process helps determine the space&#39;s volume fraction and degree of twisting.

使用离子选择性和离子电渗疗法微电极表征脑切片中的细胞外间隙

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;&#160;&#160;
1. ...