Name: homochronic transplantation of interneuron precursors into early postnatal mouse brains
Uploaded: 2018-06-08T14:30:00
Description: Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

这项研究得到了国家卫生研究院 (K99MH104595) 和发育研究院校内研究计划的支持 T.J.P。 我们感谢 Gord Fishell, 在其实验室中最初建立了这种方法。

所有实验程序均按照国家卫生研究院的指导方针进行, 并经发育研究院动物护理和使用委员会 (ACUC) 批准。下面所述的协议利用Nkx2.1 的c++;Ai9+/- 幼崽收获梅兰日兰衍生 interneuron 前体, 但可以执行任何所需的荧光记者鼠标线。雄性和雌性早产后小鼠 (P0-P2) 被不分青红皂白地用于供体和宿主组织。
1. 解决方案准备
<ol><li>根据以下食谱 (单位毫米) 制备蔗糖人...

<ol>
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该协议的一个关键方面是最大限度地提高细胞的生存能力。确保组织和细胞始终处于冰冷 carboxygenated sACSF 是促进细胞存活所必需的。这需要一个有效的解剖和离解策略, 以尽量减少细胞在不同的解决方案和大脑环境之外的时间长短。根据被解剖和移植的大脑区域的数量, 在解剖和/或移植步骤中有一个合作伙伴帮助减少实验的长度是有益的。为了保证幼犬的健康和生存, 在移植过程中尽量减少冰麻?...

适当的皮质功能需要平衡兴奋投射神经元和抑制 GABAergic 中间神经元, 一个极异质的人群, 具有明显的形态, 电生理特性, 连通性和神经化学标记。异常发育和功能的中间神经元 (和特定的 interneuron 子群) 已与病理精神障碍, 如精神分裂症, 自闭症和癫痫1,2,3。此外, 许多与这些脑部疾病相关的基因在年轻的中间神经元4中有强烈的丰富。因此, 需要更多地了解调节 interneuron 命运确定和成熟的机制, 以了解许多脑部疾病的正常发展和潜在病因。
前脑中间神经元主要来自两个瞬时胚胎结构, 内侧和尾节显赫人士 (梅兰日兰和 CGE 分别)。这些分裂后细胞 (interneuron 前体) 然后经历一个长期的切向迁移阶段, 分散在整个前脑, 在那里他们集成到广泛的电路。梅兰日兰衍生中间神经元由三个主要的非重叠的, neurochemically 定义的子群: 快速峰值 parvalbumin (PV+) 中间神经元, 非快速峰值生长抑素 (SST+) 中间神经元, 和晚峰值神经元一氧化物合酶 (nNOS+) 中间神经元, 构成海马 neurogliaform 和常春藤细胞。许多实验室已经确定了梅兰日兰中的几种机制, 将初始命运决定调整为 PV+或 SST + 中间神经元, 包括格局来说的空间梯度、interneuron 前体的出生日期和神经源性分裂的模式。5,6,7,8,9,10. 有人建议, 中间神经元最初将其区分为 "主要阶级", 然后逐渐成熟为 "决定性阶级", 因为它们与环境11互动。最近的证据表明, 一些成熟的 interneuron 亚型可能是遗传的, 因为这些细胞成为分裂后在节显赫人士, 表明早期定义的内在基因程序可能发挥比以前更大的作用赞赏12,13。然而, 如何将内在遗传程序与环境线索相互作用来驱动分化为不同的 interneuron 亚型的关键问题仍在很大程度上未被探索。
许多研究已经将胚胎梅兰日兰细胞直接移植到了不同的脑区, 以协商一致的结果, 移植细胞成熟和释放 GABA 一般抑制局部内源电路14,15, 16,17,18,19。这些有希望的观察已经产生了极大的兴趣, 使用人类诱导多潜能干细胞 (hIPSC) 衍生中间神经元治疗各种脑疾病。然而, 很少的这些研究评估, 如果这些嫁接细胞成熟的预期类型的成熟中间神经元, 一个关键组成部分, 当你思考的翻译方法。
为解决环境对 interneuron 分化和成熟的影响, 设计了一种将未成熟 interneuron 前体移植到新的脑环境中的策略, 以检查嫁接中间神经元是否采用宿主的特征。环境或保留来自捐助者环境的特征20。梅兰日兰移植不适合解决这个问题, 因为梅兰日兰含有混合种群的 interneuron 和 GABAergic 投射细胞分散在许多脑区21。如果不知道这些梅兰日兰细胞将迁移到哪里, 就无法充分评估这些移植如何受大脑环境的影响。通过在产后早期 timepoints 收割 interneuron 前体, 通过获得完成迁移并到达目标脑区的未成熟细胞来规避这个问题, 但与环境的交互作用最小。通过专注于不同脑区之间差异表达的中间神经元的特定特征, 可以确定宿主环境如何改变 interneuron 属性。本议定书概述的一般方法应适用于任何想研究年轻神经元在新环境中受到挑战时如何行为的调查人员。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7653</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium bicarbonate</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S6297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Potassium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S0751</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Calcium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Magnesium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glucose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">G7528</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sucrose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7903</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Brain Matrices</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">SA-2165</td>
			<td>Only needed if harvesting striatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fine point Dumont Forceps</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-4978</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microdissecting scissors</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-5940</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Razor blades</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12-640</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pasteur pipettes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">1367820C</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Nanoject III</td>
			<td style="width:203px;">Drummond</td>
			<td style="width:136px;">3-000-207</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Manual Manipulator w/ stand</td>
			<td style="width:203px;">World Precision Instruments&#160;</td>
			<td style="width:136px;">M3301R/M10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">5 ml round bottom plastic tubes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">149591A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">60 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12556001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">100 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12565100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pronase</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">10165921001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">16140063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DNase I</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">4716728001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Celltrics 50um filters</td>
			<td style="width:203px;">Sysmex</td>
			<td style="width:136px;">04-0042327</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypan blue</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">15-250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Hemocytometer</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">02-671-6</td>
			<td></td>
		</tr>
	</tbody>
</table>

homochronic transplantation of interneuron precursors into early postnatal mouse brains

神经元的命运决定和成熟需要一个复杂的相互作用的基因程序和环境信号。然而, 解析的作用, 内在的和外在的机制, 调节这种分化过程是一个难题, 所有发展 neurobiologists。这个问题被放大的 GABAergic 中间神经元, 一个令人难以置信的异构细胞群体出生的瞬态胚胎结构和经历一个长期的迁移阶段, 分散在整个前脑。为了探索不同的大脑环境如何影响 interneuron 的命运和成熟, 我们制定了一个协议, 以收获荧光标记未成熟的 interneuron 前体从特定脑区的新生小鼠 (P0-P2)。在这个年龄, interneuron 迁移几乎完成, 这些细胞居住在他们的最后休息环境与相对较少的突触整合。通过流式细胞仪收集单细胞溶液后, 这些 interneuron 前体被移植到 P0-P2 wildtype 产后幼崽中。通过执行伦 (如皮质对皮质) 或异位 (如皮质对海马) 移植, 你可以评估在新的大脑环境中如何挑战未成熟中间神经元影响他们的命运, 成熟和电路整合。大脑可以在成年小鼠身上收获, 并对移植细胞进行广泛的 posthoc 分析, 包括免疫组化、电生理和转录分析。这一一般方法为调查人员提供了一种策略, 用于分析不同的大脑环境如何影响神经元发育的许多方面, 并确定特定的神经元特征是否主要由硬质遗传程序驱动或环境暗示。

该协议展示了如何从产后早期大脑中采集特定的脑区 (图 1-2), 收集 interneuron 前体的单细胞 dissociations, 并将这些细胞移植到不同的脑区产后幼崽 (图 3)。在 posthoc 分析中, 接受 interneuron 前体移植的大脑在 P30-35 之间采集细胞形态学、神经化学标志物和电生理特性的特征。这些类型的化验通常是在正常小鼠的 P21-P30 之?...

Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

挑战新脑区的年轻神经元, 可以揭示环境如何造型神经元的命运和成熟的重要洞察力。本议定书描述了从特定脑区获取 interneuron 前体的程序, 并将其移植到产后幼崽的大脑中 homotopically 或 heterotopically。

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Research

JoVE Journal

Neuroscience

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

利用基于病毒的Cre重组马赛克产后小鼠脑开发基因功能分析

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

科研

神经科学

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

小鼠神经干细胞移植手术与器官的诱向性鼠肝炎病毒感染小鼠的脊髓

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Simultaneous Cryosectioning of Multiple Rodent Brains

多鼠脑的同时 Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

从产后小鼠小脑中纯化Promin-1+干细胞

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...