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>

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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 border="0" cellpadding="0" cellspacing="0" width="997">
	<tbody>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Vibratome</td>
			<td style="width:241px;">DSK</td>
			<td style="width:257px;">Microslicer Zero 1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Mouse: C57BL/6NTac inbred mice</td>
			<td>Taconic</td>
			<td style="width:257px;">Stock#B6</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Electrode puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Ag/AgCl ground pellet</td>
			<td>WPI</td>
			<td>EP2</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">pCLAMP10.3</td>
			<td style="width:241px;">Molecular Devices</td>
			<td style="width:257px;">n/a</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Custom microfil 28G tip</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">CMF28G</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Tungsten Rod</td>
			<td style="width:241px;">A-M Systems</td>
			<td style="width:257px;">716000</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Bipolar stimulating electrodes</td>
			<td style="width:241px;">FHC</td>
			<td style="width:257px;">MX21XEW(T01)</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Stimulus isolator</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">A365</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Grass S88 Stimulator</td>
			<td style="width:241px;">Grass Instruments Company</td>
			<td style="width:257px;">S88</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Borosilicate glass pipettes</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">1B150-4</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">A to D board</td>
			<td style="width:241px;">Digidata 1322A</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Signal Amplifier</td>
			<td style="width:241px;">Multiclamp 700A or 700B</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Headstage</td>
			<td style="width:241px;">CV-7B Cat 1</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Patch computer</td>
			<td style="width:241px;">Dell</td>
			<td style="width:257px;">n/a</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S5886</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Potassium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">P3911</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">HEPES</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">H3375</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Bicarbonate</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S5761</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Phosphate Monobasic</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S0751</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">D-glucose</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">G7528</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Calcium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">21108</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Magnesium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">M8266</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">valinomycin</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">V0627-10mg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">1,2-dimethyl-3-nitrobenzene</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">40870-25ml</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Potassium tetrakis (4-chlorophenyl)borate</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">60591-100mg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">5% dimethyldichlorosilane in heptane</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">85126-5ml</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">TTX</td>
			<td style="width:241px;">Cayman Chemical Company</td>
			<td style="width:257px;">14964</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Hydrochloric acid</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">H1758-500mL</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sucrose</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S9378-5kg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Pipette Micromanipulator</td>
			<td style="width:241px;">Sutter</td>
			<td style="width:257px;">MP-285 / ROE-200 / MPC-200</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Objective lens</td>
			<td style="width:241px;">Olympus</td>
			<td style="width:257px;">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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.9K 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

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

科研

神经科学

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

使用基于 FRET 的传感器 ATeam1.03YEMK在小鼠大脑的组织切片中进行细胞内 ATP 成像

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

成人脑组织切片中钾离子选择性电极的制备、检测及应用

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