Name: utilizing in vivo postnatal electroporation to study cerebellar granule neuron morphology and synapse development
Uploaded: 2021-06-09T14:30:00
Description: Watch this Scientific Journal Video about Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development at JoVE.com

这项工作得到了NIH资助R01NS098804（A.E.W.），F31NS113394（U.C.）和杜克大学夏季神经科学项目（D.G.）的支持。

注意：所有程序均根据杜克大学机构动物护理和使用委员会（IACUC）批准的协议进行。
1. 体内电穿孔或 IVE 的 DNA 制备（术前 1 天）
<ol><li>收集以下材料：纯化的DNA（每只动物0.5-25μg），3M乙酸钠，乙醇，固绿染料，超纯蒸馏水，磷酸盐缓冲溶液（PBS）（见材料表）。 注意：对于DNA，从Addgene（FUGW，https://www.addgene.org/14883/）获得在人泛素启动子下表达绿色荧光?...

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of the cerebellar system : in relation to its evolution, structure, and functions. , (1997).</li><li>Rahimi-Balaei, M., Bergen, H., Kong, J., Marzban, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+migration+during+development+of+the+cerebellum.">Neuronal migration during development of the cerebellum.</a> Frontiers in Cellular Neuroscience. 12, 484 (2018).</li><li>Alder, J., Cho, N. K., Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+precursor+cells+from+the+rhombic+lip+are+specified+to+a+cerebellar+granule+neuron+identity.">Embryonic precursor cells from the rhombic lip are specified to a cerebellar granule neuron identity.</a> Neuron. 17 (3), 389-399 (1996).</li><li>Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neural+patterning+and+specification+in+the+developing+cerebellum.">Mechanisms of neural patterning and specification in the developing cerebellum.</a> Annual Review of Neuroscience. 18, 385-408 (1995).</li><li>Ben-Arie, N., 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=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390 (6656), 169-172 (1997).</li><li>Borghesani, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+stimulates+migration+of+cerebellar+granule+cells.">BDNF stimulates migration of cerebellar granule cells.</a> Development. 129 (6), 1435-1442 (2002).</li><li>Espinosa, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+neurogenesis+and+differentiation:+insights+from+quantitative+clonal+analyses+of+cerebellar+granule+cells.">Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells.</a> Journal of Neuroscience. 28 (10), 2301-2312 (2008).</li><li>Markwalter, K. H., Yang, Y., Holy, T. E., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+coding+of+vermal+granule+neurons+in+the+developing+mammalian+cerebellum.">Sensorimotor coding of vermal granule neurons in the developing mammalian cerebellum.</a> Journal of Neuroscience. 39 (34), 6626-6643 (2019).</li><li>Shalizi, 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=PIASx+is+a+MEF2+SUMO+E3+ligase+that+promotes+postsynaptic+dendritic+morphogenesis.">PIASx is a MEF2 SUMO E3 ligase that promotes postsynaptic dendritic morphogenesis.</a> Journal of Neuroscience. 27 (37), 10037-10046 (2007).</li><li>Shalizi, 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+Calcium-regulated+MEF2+sumoylation+switch+controls+poststynaptic+differentiation.">A Calcium-regulated MEF2 sumoylation switch controls poststynaptic differentiation.</a> Science. 311 (5763), 1012-1017 (2006).</li><li>Konishi, Y., Stegmuller, J., Matsuda, T., Bonni, S., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdh1-APC+controls+axonal+growth+and+patterning+in+the+mammalian+brain.">Cdh1-APC controls axonal growth and patterning in the mammalian brain.</a> Science. 303 (5660), 1026-1030 (2004).</li><li>Holubowska, A., Mukherjee, C., Vadhvani, M., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+cerebellar+granule+neurons+in+vitro+and+in+vivo+to+study+neuronal+morphology+and+migration.">Genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration.</a> Journal of Visualized Experiments: JoVE. (85), e51070 (2014).</li><li>Yang, Y., 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=Chromatin+remodeling+inactivates+activity+genes+and+regulates+neural+coding.">Chromatin remodeling inactivates activity genes and regulates neural coding.</a> Science. 353 (6296), 300-305 (2016).</li><li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+scaling+of+cortical+and+cerebellar+numbers+of+neurons.">Coordinated scaling of cortical and cerebellar numbers of neurons.</a> Frontiers in Neuroanatomy. 4, 12 (2010).</li><li>Wilson, P. M., Fryer, R. H., Fang, Y., Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astn2,+a+novel+member+of+the+astrotactin+gene+family,+regulates+the+trafficking+of+ASTN1+during+glial-guided+neuronal+migration.">Astn2, a novel member of the astrotactin gene family, regulates the trafficking of ASTN1 during glial-guided neuronal migration.</a> Journal of Neuroscience. 30 (25), 8529-8540 (2010).</li><li>Kokubo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF-mediated+cerebellar+granule+cell+development+is+impaired+in+mice+null+for+CaMKK2+or+CaMKIV.">BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV.</a> Journal of Neuroscience. 29 (28), 8901-8913 (2009).</li><li>Schwartz, P. M., Borghesani, P. R., Levy, R. L., Pomeroy, S. L., Segal, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+cerebellar+development+and+foliation+in+BDNF-/-+mice+reveals+a+role+for+neurotrophins+in+CNS+patterning.">Abnormal cerebellar development and foliation in BDNF-/- mice reveals a role for neurotrophins in CNS patterning.</a> Neuron. 19 (2), 269-281 (1997).</li><li>Segal, R. A., Pomeroy, S. L., Stiles, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+and+fasciculation+linked+to+differential+expression+of+BDNF+and+NT3+receptors+in+developing+cerebellar+granule+cells.">Axonal growth and fasciculation linked to differential expression of BDNF and NT3 receptors in developing cerebellar granule cells.</a> Journal of Neuroscience. 15 (7), 4970-4981 (1995).</li><li>Zhou, P., 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=Polarized+signaling+endosomes+coordinate+BDNF-induced+chemotaxis+of+cerebellar+precursors.">Polarized signaling endosomes coordinate BDNF-induced chemotaxis of cerebellar precursors.</a> Neuron. 55 (1), 53-68 (2007).</li><li>Dhar, M., Hantman, A. W., Nishiyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+pattern+and+structural+factors+of+dendritic+survival+in+cerebellar+granule+cells+in+vivo.">Developmental pattern and structural factors of dendritic survival in cerebellar granule cells in vivo.</a> Scientific Reports. 8 (1), 17561 (2018).</li><li>Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+the+cerebellar+cortex+and+its+role+in+motor+learning.">Synaptic plasticity in the cerebellar cortex and its role in motor learning.</a> Canadian Journal of Neurological Sciences. 20, 70-74 (1993).</li><li>Jorntell, H., Hansel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+memories+upside+down:+bidirectional+plasticity+at+cerebellar+parallel+fiber-Purkinje+cell+synapses.">Synaptic memories upside down: bidirectional plasticity at cerebellar parallel fiber-Purkinje cell synapses.</a> Neuron. 52 (2), 227-238 (2006).</li><li>Nakanishi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+study+of+information+processing+in+the+cerebellum.">Genetic manipulation study of information processing in the cerebellum.</a> Neuroscience. 162 (3), 723-731 (2009).</li><li>Chang, C. H., 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=Atoh1+controls+primary+cilia+formation+to+allow+for+SHH-triggered+granule+neuron+progenitor+proliferation.">Atoh1 controls primary cilia formation to allow for SHH-triggered granule neuron progenitor proliferation.</a> Developmental Cell. 48 (2), 184-199 (2019).</li></ol>

小脑颗粒神经元是哺乳动物大脑中最丰富的神经元，几乎占啮齿动物大脑中神经元总数的60-70%1，14。小脑已被广泛用于阐明细胞增殖、迁移、树突形成和突触发育的机制6，9，10，11，15，16，17，18，<sup class="xref"...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62568">Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development</a>. A figure was updated.
Figure 2 was updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62568/62568fig02.jpg" /> 
Figure 2: In vivo cerebellar electroporation of granule neuron progenitors in P7 wildtype mouse pups. (A) Pups are anesthetized with 4% isoflurane delivered at a rate of 0.8L/min&#160;to ensure anesthesia throughout the injection of the DNA solution. Isoflurane is delivered at a rate of 0.8 L/min. (B) After sterilizing the mouse 3 times with betadine and 70% ethanol, an incision is made that spans the distance of the ears, revealing the hindbrain. (C) A magnified image of a white demarcation on the cranium, a landmark for the injection site. DNA construct should be injected within 1 mm above the mark; dotted lines outline the demarcation, and black arrow denotes the injection site. The ridges of the cerebellar vermis may be visible and can be useful for finding the injection site. (D) Tweezer-type electrode orientation for efficient electroporation. Plus (+) end must be oriented downwards to pull negatively charged DNA into the cerebellar parenchyma prior to administration of electrical pulses. (E) Test injection of 1 &#181;L of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62568/62568fig2v2.jpg" /> 
Figure 2: In vivo cerebellar electroporation of granule neuron progenitors in P7 wildtype mouse pups. (A) Pups are anesthetized with 4% isoflurane delivered at a rate of 0.8L/min&#160;to ensure anesthesia throughout the injection of the DNA solution. Isoflurane is delivered at a rate of 0.8 L/min. (B) After sterilizing the mouse 3 times with betadine and 70% ethanol, an incision is made that spans the distance of the ears, revealing the hindbrain. (C) A magnified image of a white demarcation on the cranium, a landmark for the injection site. DNA construct should be injected within 1 mm above the mark; dotted lines outline the demarcation, and black arrow denotes the injection site. The ridges of the cerebellar vermis may be visible and can be useful for finding the injection site. (D) Tweezer-type electrode orientation for efficient electroporation. Plus (+) end must be oriented downwards to pull negatively charged DNA into the cerebellar parenchyma prior to administration of electrical pulses. (E) Test injection of 1 &#181;L of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62568">Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development</a>. A figure was updated.

Erratum: Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

大脑发育是一个从胚胎发生到出生后生命的漫长过程。在此期间，大脑整合了内在和外在刺激的组合，这些刺激塑造了树突和轴突之间的突触连接，最终指导行为。啮齿动物小脑是研究突触如何发展的理想模型系统，因为单个神经元类型的小脑颗粒神经元（CGN）的发育可以在从祖细胞过渡到成熟神经元时进行跟踪。这部分是由于大多数小脑皮层在出生后发育，这使得出生后易于遗传操作和细胞标记1。
在哺乳动物中，CGN分化始于胚胎发育结束时，当时后脑中的增殖细胞子集迁移到菱形唇上，在小脑表面形成次级生发区2，3，4。尽管它们完全致力于颗粒神经元祖细胞（GNP）身份，但这些细胞继续在外部颗粒层（EGL）的外部增殖，直到出生后第14天（P14）。这一层的增殖导致小脑的大规模扩张，因为这些细胞只产生CGNs5。一旦新生的CGN退出EGL中的细胞周期，它们就会向内迁移到内部颗粒层（IGL），留下一个轴突，该轴突将在小脑的分子层中分叉并行进，形成平行纤维，突触到浦肯野细胞上6。这些纤维在分子层中的位置取决于细胞周期退出的时间。
首先分化的CGN将其平行纤维留向分子层的底部，而分化的CGN的轴突聚集在顶部7，8。一旦CGN细胞体到达IGL，它们就开始细化树突并与附近的抑制性和兴奋性神经元形成突触。CGN的成熟树枝状树表现出具有四个主要过程的刻板结构。在CGN成熟过程中，这些树突末端的结构形成一个爪子，该爪子富含突触后蛋白9，10。这些称为树突爪的特殊结构包含颗粒神经元上的大部分突触，对于接收来自脑桥的苔藓纤维神经支配的兴奋性输入以及来自局部高尔基体细胞的抑制性输入非常重要。一旦完全配置，CGN的突触连接允许这些细胞将输入从小脑前核中继到浦肯野细胞，浦肯野细胞从小脑皮层投射到小脑深部核。
GNPs的出生后体内电穿孔优于其他基于标记的方法，例如病毒感染和转基因小鼠系的产生，因为可以在快速的时间轴上实现所需构建体的表达，并且该方法针对少量细胞群，可用于研究细胞自主效应。该方法已在先前的研究中用于研究CGN的形态发育;然而，这些研究集中在单个时间点或短时间窗口9，10，11，12，13。该标记方法与图像分析相结合，以记录出生后前三周CGN分化的整个时间过程中发生的CGN形态变化。这些数据揭示了中广核树突发育的动态，这是小脑回路构建的基础。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Betadine</td>
			<td>Purdue Production</td>
			<td>67618-150-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Cemented 10 &#181;L needle</td>
			<td>Hamilton</td>
			<td>1701SN (80008)</td>
			<td>33 gauge, 1.27 cm (0.5 in), 4 point style</td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>Millipore Sigma</td>
			<td>AB16901</td>
			<td>Our lab uses this antibody at a 1:1000 concentration</td>
		</tr>
		<tr>
			<td>Cotton-tip applicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-chicken Cy2</td>
			<td>Jackson ImmunoResearch</td>
			<td>703-225-155</td>
			<td>Our lab uses this antibody at a 1:500 concentration</td>
		</tr>
		<tr>
			<td>Ethanol (200 proof)</td>
			<td>Koptec</td>
			<td>V1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator ECM 830</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>FUGW plasmid</td>
			<td>Addgene</td>
			<td>14883</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td>Superfrost plus</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Softheat</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342 fluorescent dye</td>
			<td>Invitrogen</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover glass</td>
			<td>VWR</td>
			<td>48382-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Sally Hansen</td>
			<td>Color 109</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Gibco</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. embedding compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus MVX10 Dissecting Scope</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipette reach tip</td>
			<td>Fisherbrand</td>
			<td>02-707-138</td>
			<td>Used for needle spacer</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (10x)</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Simple Neurite Tracer</td>
			<td>FIJI</td>
			<td></td>
			<td>https://imagej.net/Simple_Neurite_Tracer:_Basic_ 
			Instructions</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tools</td>
			<td>RWD Life Science</td>
			<td></td>
			<td>Small scissors and tweezers</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Roche</td>
			<td>11332481001</td>
			<td>non-ionic detergent</td>
		</tr>
		<tr>
			<td>Tweezertrodes</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0489</td>
			<td>5 mm, platinum plated tweezer-type electrodes</td>
		</tr>
		<tr>
			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting media</td>
			<td>Vectashield</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss 780 Upright Confocal</td>
			<td>Zeiss</td>
			<td>780</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilizing in vivo postnatal electroporation to study cerebellar granule neuron morphology and synapse development

神经元在大脑发育过程中经历其结构和功能的动态变化，以与其他细胞形成适当的连接。啮齿动物小脑是跟踪单细胞类型小脑颗粒神经元（CGN）随时间变化的发育和形态发生的理想系统。在这里，发育中的小鼠小脑中颗粒神经元祖细胞的 体内 电穿孔用于稀疏标记细胞以进行随后的形态学分析。该技术的功效体现在它能够展示CGN成熟的关键发育阶段，特别关注树突爪的形成，树突爪是这些细胞接收大部分突触输入的特殊结构。除了在整个小脑发育过程中提供CGN突触结构的快照外，该技术还可以适应以细胞自主的方式对颗粒神经元进行遗传操作，以研究任何感兴趣的基因的作用及其对CGN形态，爪发育和突触发生的影响。

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62568/62568fig04.jpg"> 图4：小脑发育过程中颗粒神经元形态分析。 （A） 从3DPI到14DPI（出生后年龄P10至P21）、细胞核（蓝色）和GFP（绿色）的电穿孔CGN的最大投影图像;箭头表示单个树突，比例尺为10μm。 （B）树突的平均数量。（C）从体细胞基部到枝晶尖端测量的平均枝晶长度。（...

Watch this Scientific Journal Video about Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development at JoVE.com

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

在这里，我们描述了一种方法，当这些细胞细化其突触结构并形成突触以整合到整个脑回路中时，在出生后大脑发育的时间过程中可视化小鼠小脑中颗粒神经元的突触发生。

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

该协议研究不同发育阶段的小脑颗粒神经元，以可视化关键的形态生长。该技术的优点包括细胞特异性靶向，转染构建体的快速表达以及细胞的稀疏标记，从而可以研究细胞自主效应。首先从加载移液器吸头上切下 11.2 毫米段，然后将切割部分放在汉密尔顿注射器的尖端作为垫片上，以将注射深度限制在 1.5 毫米。用粘合剂或封口膜将垫片固定在注射器尖端上。为了研究实验幼犬矢状脑切片的单个电穿孔 CGN 的形态，请在共聚焦显微镜上以每叠 0.5 微米的速度拍摄 Z 堆栈图像。每个图像窗口对一个单元格进行成像，以便于图像分析和3D重建。使用简单的神经突示踪剂以盲方式分析神经突长度和树突爪形成。将电穿孔CGN的单通道Z堆栈图像上传到斐济，然后单击插件“分割”和简单的神经突示踪剂“选择创建新的3D查看器”从下拉菜单中。滚动到连接到细胞体的树突的底部，然后单击连接处开始路径。通过单击单元格填充信号最亮的部分并按 Y 保留跟踪来手动跟踪路径。追踪直到枝晶的末端，然后按F确认路径。或者，追踪到爪子的底部。接下来，从结构的底部到最长的神经突的末端追踪爪子。通过按住 Windows 上的控制或 macOS 上的 ALT 并单击路径来跟踪二级和三级分支。按 F.观察迹线的测量值是否在单独的窗口中可见，以确认路径的路径。将爪子分支的所有大小相加，得到每个爪子的总长度。在代表性分析中，注射后3至14天的电穿孔CGN的投影图像显示树突数量逐渐减少。CGN经历了树枝状生长的阶段，然后从3 DPI细化到7 DPI，导致修剪了超过50%的多余树突。这一事件与剩余的乔木在每个枝晶末端形成爪状结构的逐渐延长相吻合，表明这些发育过程同时发生。到7 DPI，在大约75%的树突上发现了爪子。每个标记的CGN在NMRS中重建，以量化总体细胞 - 树枝状表面积和体积。在整个开发过程中，CGN 大小没有观察到显着差异。虽然在 7 DPI 时，CGN 的体积与 3、5 和 10 DPI 相比显着减少了 20%。手术中最重要的是在注射前准确定位小脑。该方法可以适用于体内基因的遗传操作，以通过转染shRNA，siRNA或Cre重组酶来研究它们在颗粒神经元发育中的作用。

Research

JoVE Journal

Neuroscience

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

科研

神经科学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Laser Nanosurgery of Cerebellar Axons In Vivo

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

神经肌肉接头处：测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

利用体内出生后电穿孔研究小脑颗粒神经元形态和突触发育