我们承认来自欧洲研究理事会 (FP7/260916)、亚历山大·洪堡-基金会 (Sofja Kovalevskaja) 和德意志 Forschungsgemeinschaft (DE1951 和 SFB889) 的资助。

这里描述的所有动物实验都是按照德国动物福利指南进行的。
注意: 协议的时间轴和主要步骤如图 1所示。
1. 准备工作
<ol><li>
准备以下解决方案。
	<ol>
<li>准备 DMEM + 为培养多边形 MD 星形胶质细胞: DMEM 10% FCS, 100 U 青霉素和100µg/毫升链霉素。</li>
		<li>为培养星状胶质细胞制备 NB + H: neurobasal 培养基, 5 ng/毫升 HBEGF, 1x B-27 补充, 1x 谷氨酰胺补充剂, 100 U 青霉素和100µg/毫升链霉素。 注: 等分浓度较高的 HBEGF 可在需要时在-20 ° c 下添加到培养基中。</li>
		<li>准备解剖培养基 (10 毫米 HEPES 在 HBSS)。</li>
		<li>在15毫升管中制备6-7 毫升0.05% 胰蛋白酶-edta 和6-7 毫升0.25% 胰蛋白酶-edta 的等分。这些可以被存放在-20 ° c 和解冻在37° c 水浴。</li>
	</ol>
</li>
	<li>在开始该协议的前一天, 10 厘米的组织培养处理的菜肴与5毫升的0.04% 亚 (PEI) 每一个。</li>
	<li>第二天, 去掉 PEI, 用去离子洗碗, 蒸馏水两次。在37° c 和 5% CO2中, 将12毫升的 DMEM 加到每个板和 pre-equilibrate 中, 至少在电镀电池前30分钟。</li>
	<li>准备解剖区域: 在层流解剖罩, 放置两个10厘米的培养皿, 填充在冰上的解剖介质。对于要隔离的每个脑区 (例如, 皮质, 海马), 准备一个15管, 填充14毫升解剖培养基, 并保持冰。喷雾解剖工具 (细剪刀, #3 和 #5 钳) 与70% 乙醇和地方的解剖显微镜旁边的遮光罩。</li>
</ol>2. 大鼠或小鼠脑解剖
注: 小鼠和老鼠都可以使用。年龄比 P8 的动物也可以使用 (我们测试了 P12), 但去除脑膜将变得越来越困难。此外, 细胞的产量和健康将会减少, 因为来自较年长动物的脑细胞将形成复杂的过程和连接, 在组织分离过程中可能破裂, 导致细胞死亡增加。皮质制剂在这里描述, 但其他大脑区域也可以使用。
<ol><li>在37° c 水浴中放置0.05% 胰蛋白酶-EDTA。</li>
	<li>安乐动物 (为 E19 幼崽, 或 P0-P8 动物) 通过缓慢提高 CO2浓度, 并隔离大脑。以下步骤需要无菌技术。</li>
	<li>
如果使用胚胎组织, 首先将胚胎从子宫中取出, 然后打开胚胎囊, 然后立即用剪刀切断头部。当所有的头被切断, 转移到预解剖介质。
	<ol>
<li>一旦头部被淹没在介质中, 打开皮肤和头骨 (两个都是非常柔软的幼小动物) 与钳子和掐掉小脑。小心地把大脑的其余部分从头骨上抬起来。 注: 两到四 E19 胚胎在解剖当天为每道50万细胞的皮层星形胶质细胞至少八10厘米的食物提供足够的组织。</li>
	</ol>
</li>
	<li>对于新生儿或年长的幼崽, 用剪刀切断头部, 通过内侧从头部后部到鼻尖的一条直线切割皮肤来隔离大脑。把皮肤拉到一边, 小心地把头骨向左和向右, 然后抬起头骨。切断小脑, 并把大脑的其余部分从头骨中取出。 注: 两到三 P0-P8 幼崽的组织至少八10厘米的皮质星形胶质细胞在解剖的时候每道50万细胞上都有一组。</li>
	<li>将大脑皮层与底层的中脑结构分开。把镊子放在两半球之间的纵裂处, 然后将镊子移到一个半球的皮质和中脑结构之间 (垂直于纵裂), 以释放每个半球。</li>
	<li>使用镊子, 从每个半球 (及其海马) 中取出所有脑膜, 然后分离海马。如果需要海马星形胶质细胞, 将每个海马体移动到一个15毫升的管内, 在冰上填满14毫升的解剖介质。</li>
	<li>如果需要特定区域 (例如, 额叶皮质), 则从每个半球的皮层移除更多区域。切割任何皮质组织, 用于将星形胶质细胞生成不大于 1 mm3的碎片。收集所有的皮质组织在一个单独的15毫升的管中填充解剖介质冰。</li>
</ol>3. 组织消化和分离
<ol><li>一旦所有的组织碎片收集, 等待他们定居到底部的管, 然后吸尽可能多的培养基。</li>
	<li>用血清学吸管加2-3 毫升0.05% 胰蛋白酶-EDTA, 在37° c 的水浴中孵育20分钟的试管。 注: 将所有胰蛋白酶-EDTA 从吸管中迅速释放, 混合组织块并允许再次沉淀。</li>
	<li>小心地抽出胰蛋白酶-EDTA, 将组织块留在尽可能少的液体中, 在试管的底部。</li>
	<li>加入5毫升的预剥离培养基, 冲洗剩余的胰蛋白酶-EDTA。吸管在管子上部的一侧, 以达到混合的组织件。</li>
	<li>吸取解剖介质, 并尽可能少地将组织块留在液体中。重复这个洗涤步骤与预解剖介质两次以上。</li>
	<li>最后的洗涤步骤后, 添加1毫升的 DMEM + (温暖到室温) 使用1000µL 吸管尖端, 研制的组织由移上下不引入气泡。 注意: 避免产生气泡是很重要的, 因为这可能会改变溶液的 pH 值, 而星形胶质细胞是非常敏感的。</li>
	<li>在50毫升管的开口处放置一个100µm 的细胞过滤器, 并湿4.5 毫升的预 DMEM。</li>
	<li>将1毫升的游离组织悬浮液移到细胞过滤器上, 再添加4.5 毫升预 DMEM, 通过细胞过滤器清洗细胞。</li>
</ol>4. 传代星形胶质细胞富集的电池电镀
<ol><li>计算细胞,例如, 通过稀释10µL 的细胞悬液与10µL 台盼蓝和移混合到例。</li>
	<li>板50万细胞在12毫升 DMEM + 每10厘米的菜。 注: DMEM + 有助于星的生长, 比神经元或小胶质细胞更重要。在摇晃过程中, 在井内的玻璃片的物理力比在10厘米的盘子上的作用要强得多 (没有片)。如果细胞在24井板上震动, 它们就会变得不健康或死亡。只有板星形胶质细胞在 24-井板震动后。</li>
	<li>保持细胞培养在孵化器在37° c 和 5% CO2七天。</li>
	<li>在6天, 在震动和传代细胞之前的前一天, 准备在步骤5中描述的 PEI 涂层培养基。</li>
</ol>5. 传代细胞
<ol><li>
前一天传代, 外套细胞培养板材与 0.04% PEI。
	<ol>
<li>对于免疫细胞化学, 病毒转导, 或质粒转染, 使用24井或6井板 (每一个包含一个灭菌12毫米或25毫米玻璃片, 分别)。</li>
		<li>将70% 乙醇中的玻璃片消毒至少1小时, 然后在去离子、蒸馏水中清洗三次, 然后将片放入井中。</li>
		<li>增加至少70µL 0.04% pei 每12毫米片在24井板材和600µL 0.04% pei 每25毫米片在 6-井板材。</li>
		<li>对于西方的印迹或 RNA 序列分析, 10 厘米的菜肴已经包含了细胞解剖后可以继续使用。盘子只需要摇晃, 然后用 1x PBS 洗净, 然后用 NB + H (不要转移到新的菜肴) 处理。</li>
	</ol>
</li>
	<li>在天体外(DIV) 7 在解剖后, 将10厘米的碗碟与细胞在 DMEM + 在孵化器的实验室振动筛和动摇的菜在 110 rpm 为6小时。</li>
	<li>用去离子、蒸馏水两次冲洗片。将 NB + H 介质添加到板上 (每井500µL 24 井板和6井板2毫升)。预平衡的板在孵化器在37° c 和 5% CO2 , 而 10 cm 菜是震动, 或至少30分钟。</li>
	<li>20-30 分钟前 6 h 震动, 预热 1x PBS, DMEM +, NB + h, 和0.25% 胰蛋白酶-EDTA 到37° c 在水浴。</li>
	<li>经过6小时的震动, 采取了培养皿关闭振动筛和立即取代培养基 (包括 shaken-off 神经元, 突和小胶质细胞) 与10毫升 5ml 1x PBS 每道菜。 注意: 重要的是要立即吸取旧培养基中含有分离细胞, 以避免 non-astrocytic 细胞重新。</li>
	<li>取出 PBS, 加入3毫升5ml 胰蛋白酶-EDTA 每道菜。孵育每道菜4分钟在孵化器在37° c 和 5% CO2。 注: 如果准备样品为西方印迹或 RNA-Seq, 不添加胰蛋白酶-EDTA。取而代之的是, 用12毫升5ml 的铌 + H 取代 PBS, 然后将培养基放回孵化器中。</li>
	<li>在每道菜中加入5毫升 5ml DMEM + 3 毫升胰蛋白酶-EDTA, 用5毫升的血清吸管将细胞从盘子中移出, 并在50毫升管中收集细胞悬浮液。 注: 从盘子底部直接吸管--当细胞分离时, 盘子的地板会变得更清晰。只使用含血清的培养基 (代替 DMEM +), 作为血清流感胰蛋白酶。</li>
	<li>离心的细胞悬浮在 3220 x g 在20° c 为 4 min。</li>
	<li>取出上清和重1毫升5ml 的细胞颗粒。</li>
</ol>6. 最后的星形胶质细胞培养
<ol><li>计算单元格数。</li>
	<li>在6井板 (2 毫升铌 + h 介质) 或24井板 (500 µL 铌 + h 介质) 中, 每井板2万细胞。</li>
	<li>保持细胞培养在孵化器在37° c 和 5% CO2直到进一步治疗或分析。</li>
	<li>每周交换一半的培养基。 注意: 在 low-nutrient 的环境中, 星形胶质细胞停止增殖, 但任何残留的小胶质都会增殖 (尽管在我们的经验中, AWESAM 的培养中没有小胶质)。每周交换一半培养基, 可防止任何可能残留的小胶质细胞的富集8, 单一保持健康至少三周。</li>
</ol>

<ol>
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作者没有什么可透露的。

四步在协议之内是重要的: 1) 准备 HBEGF 浓度精确地 5 ng/毫升, 因为小增量在集中可能导致不成熟的文化,例如, 10 ng/ml HBEGF 将 de-differentiate 星形胶质细胞4;2) 在含有组织或细胞的溶液中避免气泡, 这会改变 pH 值, 阻碍星形胶质星的健康;3) pre-equilibrating 培养基, 达到生长健康的星形胶质细胞的最佳 pH 值和温度;4) 每周只交换一半培养基, 因为任何残留的小胶质细胞在存在足够的养...

星形胶质细胞通过营养支持、血流、突触信号和可塑性以及细胞间通讯来影响大脑功能, 所有这些都是围绕着薄星过程的机制。这里描述的 AWESAM 协议允许对这些过程的研究, 不受神经元和其他神经胶质的干扰, 这是有用的例如, 因为许多蛋白质的表达, 甚至 Ca2 +信号重叠在不同的脑细胞类型.此外, 此方法克服了以前可用技术的局限性。特别是, 我们的协议提供了在体内样的形态学 (星形胶质细胞与薄的过程) 和基因表达快速, 方便, 便宜的方式。
细胞形态、基因表达以及许多其他的调控过程都受到环境的直接影响。在培养皿中, 这指的是周围细胞释放的因子, 也是细胞生长的媒介。对于星形胶质细胞, HBEGF 以前报告诱导一种星状形态学1 , 但也发现 de-differentiate 星形胶质细胞2。然而, 较低浓度的 HBEGF 后来用于产生更多的在体内样星形胶质细胞通过 RNA 测序在两个不同的协议3,4。此外, 星形胶质细胞的形态随培养基成分的变化而改变, 不管是什么协议4: 低浓度 HBEGF 的 Neurobasal 培养基都是生长中的正晶状胶质星, 而其他培养基成分 (如如,含血清的 DMEM) 产生多边形细胞 (即使是在 immunopanning 新分离的星形胶质)。
AWESAM 协议克服了以前技术的缺点生长星形胶质细胞单一4。以前的技术为生长星形胶质细胞单一有以下缺点: 多边形形态学, 这是不典型的在体状胶质星型,在体内(MD 方法)5;协议的长度和成本 (从诱导多能干细胞制备星形胶质细胞需要三月, 需要许多试剂, 包括昂贵的生长因子)6;低量的材料 (immunopanning 方法)3;星形胶质细胞的 de-differentiation 和3D 矩阵的要求 (Puschmann et al)1,2. 相比之下, AWESAM 协议提供:在体内样的形态学 (星形胶质细胞与薄的过程);快速、简便、廉价的方法;大量的材料;与 immunopanned 和 MD 星形胶质细胞相比, 最体内样的基因表达。此外, 2D 文化允许研究的 Ca2 +信号和囊泡回收在薄的过程中, 并在事件接近膜 (例如, TIRF 显微镜是不可能在3D 文化)。
MD 方法自 1980年5发布以来已被广泛使用, 为多边形星形胶质细胞单一提供了一种简单快捷的技术。简言之, MD 方法需要在胎儿小牛血清 (FCS) 含有 DMEM 的混合脑细胞, 其次是为星形胶质细胞提供丰富的震动步骤 (就像所有其他的脑单元类型从碟中分离出来) 和在同一培养基中的进一步培养。DMEM 补充与 FCS 是用于许多其他细胞类型, 从成纤维细胞和脂肪细胞到不同的癌细胞系, 所有这些共享的多边形形态学显示由 MD 星形胶质细胞在文化。直到早期的 2000s7, 几乎没有考虑到适合于星形胶质细胞的介质, 特别是有利于其典型的星状形态学发现的体内。2011年发布的一项协议确实实现了这种星状形态学: 称为 immunopanning 方法, 它采用了最优化的无血清培养基培养星形胶质细胞3。使用这种方法, 新的孤立的脑细胞暴露在一系列的菜肴的抗体, 靶向细胞类型特定的细胞表面蛋白, 以丰富的星形胶质。尽管 immunopanned 星形胶质细胞的形态学和表达谱有更多的在体内, 但大多数的体外研究仍依赖于 MD 型胶质蛋白的方法。MD 方法是简单和快速的, 而 immunopanning 方法包括更复杂和耗时的步骤在第一天的文化 (如较长的酶消化周期, 仔细分层的解决方案的不同密度, immunopanning 本身,几个离心步骤-所有之前电镀)。但是, 通过使用更专门的介质, AWESAM 方法提供了 MD 方法的速度和 immunopanning 协议中的在体内样貌。
总的来说, AWESAM 协议是有用的研究更多的在体内状的星形胶质细胞在2D 单一从神经元和其他神经胶质分离 (如以前的特点4由 immunostainings, immunoblots, 和 RNA 测序)。它允许研究薄的星过程, 并为可视化自发的 Ca2 +信号和接近膜的事件提供了很大的可访问性 (如, 由 TIRF 显微镜成像)。

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">0.05% trypsin-EDTA</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:344px;">25300054</td>
			<td style="width:167px;">warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>100X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin / streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>50 stock; penicillin: 5000 U/ml; streptomycin: 5000 &#181;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>without L-glutamine</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBEGF</td>
			<td>Sigma</td>
			<td>4643</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBSS</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FCS</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td>1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100 &#956;m nylon cell strainer</td>
			<td>BD</td>
			<td style="width:344px;">352360</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hera Cell 240i cell culture incubator</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laboratory shaker</td>
			<td>Heidolph</td>
			<td>Rotamax 120&#160;</td>
			<td>use inside cell culture incubator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol

AWESAM (a low-成本easy stellate strocyte m方法) 协议需要一种快速、简单且廉价的方法来生成大量的在体内像老鼠和大鼠星形胶质细胞单一: 脑细胞可以从不同的脑区分离出来, 经过一周的细胞培养, non-astrocytic 细胞通过将培养皿放在孵化器中6小时的振动器上而被动摇。其余的星形胶质细胞被传代成新的板块, 并有一个特定的特殊介质 (称为 NB + H)。含低浓度肝素结合 EGF 样生长因子 (HBEGF), 用于代替血清在培养基中。AWESAM 星形胶质细胞生长于 NB + H 后, 具有星状形态, 并具有优良的工艺特点。此外, 这些星形胶质细胞比以前发表的方法产生的星形胶质细胞有更多的体内样的基因表达。Ca2 +成像, 囊泡动力学, 和其他事件接近的膜可以因此研究的精细星过程体外,例如, 使用活细胞共聚焦或 TIRF 显微镜。值得注意的是, AWESAM 星形胶质细胞也表现出类似于星形胶质细胞体内的自发 Ca2 +信号。

星形胶质细胞与神经元共, 生长在 NB + 培养基上, 在2周的培养后呈星状 (图 2)。除了铌 + 培养基, 共星形胶质细胞还暴露在未知的神经元衍生因素, 可能有助于他们的生存和形态学。相比之下, MD 星形胶质细胞生长在 DMEM + 作为单一 (在摆脱其他细胞类型之前, 通过) 出现多边形后2周。AWESAM 星形胶质细胞在震动后生长, 在2周的培养后出现星状与许多精细?...

Watch this Scientific Journal Video about Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol at JoVE.com

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

这里描述的 AWESAM 协议是最理想的培养小鼠星形胶质细胞与其他脑神经干细胞的快速, 简单, 便宜的方式。AWESAM 星形胶质细胞展示自发 Ca2 +信号, 形态学和基因表达谱类似于星形胶质细胞在体内。

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

culturing-vivo-like-murine-astrocytes-using-fast-simple-inexpensive

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

8.3K 次观看.  University of Oxford. 这里描述的 AWESAM 协议是最理想的培养小鼠星形胶质细胞与其他脑神经干细胞的快速, 简单, 便宜的方式。AWESAM 星形胶质细胞展示自发 Ca2 +信号, 形态学和基因表达谱类似于星形胶质细胞在体内。

视频: Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

科研

神经科学

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

耦合星形胶质细胞网络的大小、形状和方向性分析

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

An Emerging Target Paradigm to Evoke Fast Visuomotor Responses on Human Upper Limb Muscles

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, and size are processed and integrated within numerous brain areas, then ultimately relayed to the motor periphery. In some instances, a reaction is needed as fast as possible. These fast visuomotor transformations, and their underlying neurological substrates, are poorly understood in humans as they have lacked a reliable biomarker. Stimulus-locked responses (SLRs) are short latency (&#60;100 ms) bursts of electromyographic (EMG) activity representing the first wave of muscle recruitment influenced by visual stimulus presentation. SLRs provide a quantifiable output of rapid visuomotor transformations, but SLRs have not been consistently observed in all subjects in past studies. Here we describe a new, behavioral paradigm featuring the sudden emergence of a moving target below an obstacle that consistently evokes robust SLRs. Human participants generated visually guided reaches toward or away from the emerging target using a robotic manipulandum while surface electrodes recorded EMG activity from the pectoralis major muscle. In comparison to previous studies that investigated SLRs using static stimuli, the SLRs evoked with this emerging target paradigm were larger, evolved earlier, and were present in all participants. Reach reaction times (RTs) were also expedited in the emerging target paradigm. This paradigm affords numerous opportunities for modification that could permit systematic study of the impact of various sensory, cognitive, and motor manipulations on fast visuomotor responses. Overall, our results demonstrate that an emerging target paradigm is capable of consistently and robustly evoking activity within a fast visuomotor system.

Presented here is a behavioral paradigm that elicits robust fast visuomotor responses on human upper limb muscles during visually guided reaches.

唤起人类上肢肌肉快速视觉运动反应的新兴目标范式

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

使用遗传编码的Ca2 + 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca2 + 摄取进行成像

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

摘要

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此视频中的章节

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

测定冷灵敏度和适应小鼠一种简单廉价的方法

协议星形胶质细胞的三维共聚焦形态分析

一个简单的方法来衡量在改建悬赏寻求行为使用

一个简单的一步解剖协议成人全安装准备

耦合星形胶质细胞网络的大小、形状和方向性分析

唤起人类上肢肌肉快速视觉运动反应的新兴目标范式

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

使用遗传编码的Ca^{2 +} 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca^{2 +} 摄取进行成像

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

摘要

探索更多视频

此视频中的章节

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

测定冷灵敏度和适应小鼠一种简单廉价的方法

协议星形胶质细胞的三维共聚焦形态分析

一个简单的方法来衡量在改建悬赏寻求行为使用

一个简单的一步解剖协议成人全安装准备

耦合星形胶质细胞网络的大小、形状和方向性分析

唤起人类上肢肌肉快速视觉运动反应的新兴目标范式

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

使用遗传编码的Ca2 + 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca2 + 摄取进行成像

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合

使用遗传编码的Ca^{2 +} 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca^{2 +} 摄取进行成像