Name: dissecting cell-autonomous function of fragile x mental retardation protein in an auditory circuit by in ovo electroporation
Uploaded: 2022-07-06T15:00:00
Description: Watch this Scientific Journal Video about Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation at JoVE.com

本研究得到以下资助：国家自然科学基金（第32000697号）;广州市科技计划（202102080139）;广东省自然科学基金（2019A1515110625，2021A1515010619）;中央高校基本科研业务费专项资金（11620324）;暨南大学再生医学教育部重点实验室研究经费（No.ZSYXM202107）;中央高校基本科研业务费专项资金（21621054）;中国广东省医学科学研究基金（20191118142729581）。感谢暨南大学医学实验中心。我们感谢Terra Bradley博士对手稿的精心编辑。

鸡蛋和鸡胚胎按照暨南大学动物护理和使用委员会批准的动物规程进行谨慎和尊重的处理。
1. 卵子和质粒制备
<ol><li>鸡蛋准备<ol><li>从华南农业大学获得新鲜受精鸡蛋（Gallus gallus），并在孵化前储存在16°C。为了获得最佳的活力，请在到达后一周内设置所有种蛋进行孵化。</li>
		<li>将鸡蛋水平放置并在38°C下孵育46-48小时，直到汉堡和汉密尔顿（HH）阶段12...

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+FMR1+genotype+influences+the+degree+of+synaptic+connectivity+in+a+mosaic+mouse+model+of+fragile+X+syndrome.">Presynaptic FMR1 genotype influences the degree of synaptic connectivity in a mosaic mouse model of fragile X syndrome.</a> Journal of Neuroscience. 27 (15), 4014-4018 (2007).</li><li>Deng, P. Y., Sojka, D., Klyachko, V. 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+presynaptic+short-term+plasticity+and+information+processing+in+a+mouse+model+of+fragile+X+syndrome.">Abnormal presynaptic short-term plasticity and information processing in a mouse model of fragile X syndrome.</a> Journal of Neuroscience. 31 (30), 10971-10982 (2011).</li><li>Patel, A. B., Hays, S. A., Bureau, I., Huber, K. M., Gibson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+target+cell-specific+role+for+presynaptic+Fmr1+in+regulating+glutamate+release+onto+neocortical+fast-spiking+inhibitory+neurons.">A target cell-specific role for presynaptic Fmr1 in regulating glutamate release onto neocortical fast-spiking inhibitory neurons.</a> Journal of Neuroscience. 33 (6), 2593-2604 (2013).</li><li>Patel, A. B., Loerwald, K. W., Huber, K. M., Gibson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+FMRP+promotes+the+pruning+of+cell-to-cell+connections+among+pyramidal+neurons+in+the+L5A+neocortical+network.">Postsynaptic FMRP promotes the pruning of cell-to-cell connections among pyramidal neurons in the L5A neocortical network.</a> Journal of Neuroscience. 34 (9), 3413-3418 (2014).</li><li>Higashimori, 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=Selective+deletion+of+astroglial+FMRP+dysregulates+glutamate+transporter+GLT1+and+contributes+to+Fragile+X+syndrome+phenotypes+in+vivo.">Selective deletion of astroglial FMRP dysregulates glutamate transporter GLT1 and contributes to Fragile X syndrome phenotypes in vivo.</a> Journal of Neuroscience. 36 (27), 7079-7094 (2016).</li><li>Hodges, J. 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=Astrocytic+contributions+to+synaptic+and+learning+abnormalities+in+a+mouse+model+of+Fragile+X+syndrome.">Astrocytic contributions to synaptic and learning abnormalities in a mouse model of Fragile X syndrome.</a> Biological Psychiatry. 82 (2), 139-149 (2017).</li><li>Gonzalez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Audiogenic+seizures+in+the+Fmr1+knock-out+mouse+are+induced+by+Fmr1+deletion+in+subcortical,+VGlut2-expressing+excitatory+neurons+and+require+deletion+in+the+inferior+colliculus.">Audiogenic seizures in the Fmr1 knock-out mouse are induced by Fmr1 deletion in subcortical, VGlut2-expressing excitatory neurons and require deletion in the inferior colliculus.</a> Journal of Neuroscience. 39 (49), 9852-9863 (2019).</li><li>Bland, K. 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=FMRP+regulates+the+subcellular+distribution+of+cortical+dendritic+spine+density+in+a+non-cell-autonomous+manner.">FMRP regulates the subcellular distribution of cortical dendritic spine density in a non-cell-autonomous manner.</a> Neurobiology of Disease. 150, 105253 (2021).</li><li>Pfeiffer, B. E., Huber, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+mental+retardation+protein+induces+synapse+loss+through+acute+postsynaptic+translational+regulation.">Fragile X mental retardation protein induces synapse loss through acute postsynaptic translational regulation.</a> Journal of Neuroscience. 27 (12), 3120-3130 (2007).</li><li>Pfeiffer, B. E., 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=Fragile+X+mental+retardation+protein+is+required+for+synapse+elimination+by+the+activity-dependent+transcription+factor+MEF2.">Fragile X mental retardation protein is required for synapse elimination by the activity-dependent transcription factor MEF2.</a> Neuron. 66 (2), 191-197 (2010).</li><li>Deng, P. 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=FMRP+regulates+neurotransmitter+release+and+synaptic+information+transmission+by+modulating+action+potential+duration+via+BK+channels.">FMRP regulates neurotransmitter release and synaptic information transmission by modulating action potential duration via BK channels.</a> Neuron. 77 (4), 696-711 (2013).</li><li>Yang, Y. 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=Identification+of+a+molecular+locus+for+normalizing+dysregulated+GABA+release+from+interneurons+in+the+Fragile+X+brain.">Identification of a molecular locus for normalizing dysregulated GABA release from interneurons in the Fragile X brain.</a> Molecular Psychiatry. 25 (9), 2017-2035 (2020).</li><li>Razak, K. A., Dominick, K. C., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+studies+in+fragile+X+syndrome.">Developmental studies in fragile X syndrome.</a> Journal of Neurodevelopmental Disorders. 12 (1), 13 (2020).</li><li>Wang, X., Zorio, D. A. R., Schecterson, L., Lu, Y., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+FMRP+regulates+synaptogenesis+in+vivo+in+the+developing+cochlear+nucleus.">Postsynaptic FMRP regulates synaptogenesis in vivo in the developing cochlear nucleus.</a> Journal of Neuroscience. 38 (29), 6445-6460 (2018).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal-specific+roles+of+fragile+X+mental+retardation+protein+in+the+development+of+the+hindbrain+auditory+circuit.">Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit.</a> Development. 147 (21), (2020).</li><li>Rubel, E. W., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+system+development:+primary+auditory+neurons+and+their+targets.">Auditory system development: primary auditory neurons and their targets.</a> Annual Review of Neuroscience. 25, 51-101 (2002).</li><li>Cramer, K. S., Fraser, S. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origins+of+auditory+brain-stem+nuclei+in+the+chick+hindbrain.">Embryonic origins of auditory brain-stem nuclei in the chick hindbrain.</a> Developmental Biology. 224 (2), 138-151 (2000).</li><li>Chervenak, A. P., Hakim, I. S., Barald, K. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporation+in+the+chicken+auditory+brainstem.">In ovo electroporation in the chicken auditory brainstem.</a> Journal of Visualized Experiments. (124), e56628 (2017).</li><li>Evsen, L., Doetzlhofer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+the+chicken+auditory+organ+by+in+ovo+micro-electroporation.">Gene transfer into the chicken auditory organ by in ovo micro-electroporation.</a> Journal of Visualized Experiments. (110), e53864 (2016).</li><li>Schecterson, L. C., Sanchez, J. T., Rubel, E. W., Bothwell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrkB+downregulation+is+required+for+dendrite+retraction+in+developing+neurons+of+chicken+nucleus+magnocellularis.">TrkB downregulation is required for dendrite retraction in developing neurons of chicken nucleus magnocellularis.</a> Journal of Neuroscience. 32 (40), 14000-14009 (2012).</li><li>Wang, X. 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=High+glucose+environment+inhibits+cranial+neural+crest+survival+by+activating+excessive+autophagy+in+the+chick+embryo.">High glucose environment inhibits cranial neural crest survival by activating excessive autophagy in the chick embryo.</a> Scientific Reports. 5, 18321 (2015).</li><li>Yu, X., Wang, X., Sakano, H., Zorio, D. A. R., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+fragile+X+mental+retardation+protein+correlates+with+cellular+and+synaptic+properties+in+primary+auditory+neurons+following+afferent+deprivation.">Dynamics of the fragile X mental retardation protein correlates with cellular and synaptic properties in primary auditory neurons following afferent deprivation.</a> Journal of Comparative Neurology. 529 (3), 481-500 (2021).</li><li>Li, 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=Islet-1+expression+in+the+developing+chicken+inner+ear.">Islet-1 expression in the developing chicken inner ear.</a> Journal of Comparative Neurology. 477 (1), 1-10 (2004).</li><li>Carr, C. E., Boudreau, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+projections+of+auditory+nerve+fibers+in+the+barn+owl.">Central projections of auditory nerve fibers in the barn owl.</a> Journal of Comparative Neurology. 314 (2), 306-318 (1991).</li><li>Sandell, L. L., Butler Tjaden, N. E., Barlow, A. J., Trainor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochleovestibular+nerve+development+is+integrated+with+migratory+neural+crest+cells.">Cochleovestibular nerve development is integrated with migratory neural crest cells.</a> Developmental Biology. 385 (2), 200-210 (2014).</li><li>Cramer, K. S., Bermingham-McDonogh, O., Krull, C. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphA4+signaling+promotes+axon+segregation+in+the+developing+auditory+system.">EphA4 signaling promotes axon segregation in the developing auditory system.</a> Developmental Biology. 269 (1), 26-35 (2004).</li><li>Evsen, L., Sugahara, S., Uchikawa, M., Kondoh, H., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+neurogenesis+in+the+inner+ear+requires+inhibition+of+Sox2+transcription+by+neurogenin1+and+neurod1.">Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1.</a> Journal of Neuroscience. 33 (9), 3879-3890 (2013).</li><li>Curnow, E., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+animal+models+for+understanding+FMRP+functions+and+FXS+pathology.">New animal models for understanding FMRP functions and FXS pathology.</a> Cells. 11 (10), 1628 (2022).</li></ol>

为了确定FMRP的细胞自主功能，需要操纵其在单个细胞组或细胞类型中的表达。由于FMRP的主要功能之一是调节突触形成和可塑性，因此选择性地操纵某个回路的每个突触组件是充分了解突触通信中FMRP机制的先决条件。在鸡胚胎的卵电穿孔中，我们展示了一种在突触前AG神经元或突触后NM神经元中靶向AG-NM回路中FMRP表达的方法。为了实现这一目标，为电穿孔选择合适的...

脆性 X 综合征 （FXS） 是一种以智力障碍、感觉异常和自闭症行为为特征的神经发育障碍。在大多数情况下，FXS是由从早期胚胎阶段开始的脆性X智力迟钝蛋白（FMRP;由Fmr1基因编码）的全球损失引起的1。FMRP是一种RNA结合蛋白，通常在大脑中的大多数神经元和神经胶质细胞以及感觉器官中表达2，3，4。在哺乳动物的大脑中，FMRP可能与数百种编码对各种神经活动很重要的蛋白质的mRNA有关5。对常规Fmr1基因敲除动物的研究表明，FMRP表达对突触神经传递的组装和可塑性尤为重要6。一些条件和马赛克敲除模型进一步证明，FMRP作用和信号在几个发育事件中因大脑区域，细胞类型和突触部位而异，包括轴突投影，树突状图案和突触可塑性7，8，9，10，11，12，13，14.通过在脑切片或培养的神经元中细胞内递送抑制性FMRP抗体或FMRP本身来研究FMRP在调节突触传递方面的急性功能15，16，17，18。然而，这些方法不能在开发过程中跟踪FMRP误表达诱导的后果。因此，非常需要开发体内方法来研究FMRP的细胞自主功能，并有望帮助确定FXS患者中报告的异常是相关神经元和回路中FMRP丢失的直接后果，还是发育过程中网络范围变化的继发性后果19。
鸡胚胎的听觉脑干为电路和突触发育中FMRP调节的深入功能分析提供了一个独特的优势模型。易于获得胚胎鸡脑和成熟的用于遗传操作的 卵 电穿孔技术极大地促进了我们对早期胚胎阶段大脑发育的理解。在最近发表的一项研究中，该技术与先进的分子工具相结合，可以对FMRP错误表达进行时间控制20，21。在这里，该方法被推进到分别诱导突触前和突触后神经元的选择性操作。这种方法是在听觉脑干回路中开发的。声音信号由听觉内耳中的毛细胞检测到，然后传递到听神经节（AG;在哺乳动物中也称为螺旋神经节）。AG中的双极神经元通过其外周过程支配毛细胞，进而将中枢投影（听觉神经）发送到脑干，在那里它们终止于两个初级耳蜗核，即大细胞核（NM）和角核（NA）。NM中的神经元在结构和功能上与哺乳动物前腹侧耳蜗核的球形灌丛细胞相当。在NM内，听觉神经纤维（ANF） 突触通过 Hold终端22的大端球在NM神经元的躯体上突触。在发育过程中，NM 神经元起源于后脑23 中的菱形 5 和 6 （r5/6），而 AG 神经元源自耳囊24 中的神经母细胞。在这里，我们分别描述了在突触前AG神经元和突触后NM神经元中选择性敲低FMRP表达的过程。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>Egg incubation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16 &#176;C refrigerator</td>
			<td>MAGAT</td>
			<td></td>
			<td>Used for fertilized egg storage.</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>SHANGHAI BOXUN</td>
			<td>GZX-9240MBE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertilized eggs</td>
			<td>Farm of South China Agricultural University</td>
			<td></td>
			<td>Eggs must be used in one week for optimal viability.</td>
		</tr>
		<tr>
			<td>Plasmid preparation</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Centrifuge</td>
			<td>Sigma</td>
			<td>10016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Solarbio</td>
			<td>G1661</td>
			<td>Make 0.1% working solution in distilled water and autoclave.</td>
		</tr>
		<tr>
			<td>Plasmid Maxi-prep kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td>Dissolve plasmid DNA in Tris-EDTA (TE) buffer; endotoxin-free preparation kit</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td>Make 7.5M working solution in nuclase-free water.</td>
		</tr>
		<tr>
			<td>Electroporation and Doxycycline Administration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>BTX</td>
			<td>ECM399</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL / 5 mL Syringe</td>
			<td>GUANGZHOU KANGFULAI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>CNOPTEC</td>
			<td>SZM-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxcycline</td>
			<td>Sigma-Aldrich</td>
			<td>D9891</td>
			<td>Use fresh aliquots for each dose and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>BEIBOBOMEI</td>
			<td>RD0910</td>
			<td>0.9-1.1 mm*100 mm</td>
		</tr>
		<tr>
			<td>Laboratory parafilm</td>
			<td>PARAFILM</td>
			<td>PM996</td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>CHENGDU INSTRUMENT FACTORY</td>
			<td>WD-2</td>
			<td>Pulling condition: 500 &#176;C for 15 s</td>
		</tr>
		<tr>
			<td>Platinum elctrodes</td>
			<td>Home made</td>
			<td></td>
			<td>0.5 mm diameter, 1.5 mm interval.</td>
		</tr>
		<tr>
			<td>Platinum elctrodes</td>
			<td>Home made</td>
			<td></td>
			<td>0.5 mm diameter, 1.5 mm interval.</td>
		</tr>
		<tr>
			<td>Rubber tube</td>
			<td>Sigma-Aldrich</td>
			<td>A5177</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Dissection and Fixation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>RWD</td>
			<td>F11020-11</td>
			<td>Tip size: 0.05*0.01 mm</td>
		</tr>
		<tr>
			<td>Other surgery tools</td>
			<td>RWD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>Freshly made 4% PFA solution in phosphate-buffered saline can be stored in 4 &#176;C for up to 1 week.</td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>DOW</td>
			<td>01673921</td>
			<td>For black background plates, food-grade carbon powder is applied.</td>
		</tr>
		<tr>
			<td>Sectioning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>LEICA</td>
			<td>CM1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td>From bovine skin.</td>
		</tr>
		<tr>
			<td>Sliding microtome</td>
			<td>LEICA</td>
			<td>SM2010</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostaining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-Mouse</td>
			<td>Abcam</td>
			<td>ab150113</td>
			<td>1:500 dilution, RRID: AB_2576208</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti-rabbit</td>
			<td>Abcam</td>
			<td>ab150078</td>
			<td>1:500 dilution, RRID: AB_2722519</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abcam</td>
			<td>ab285390</td>
			<td>1: 1000 dilution</td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting medium</td>
			<td>Southern&#160;Biotech</td>
			<td>Sb-0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>FMRP antibody</td>
			<td>Y. Wang, Florida State University</td>
			<td>#8263</td>
			<td>1:1000 dilution, RRID: AB_2861242</td>
		</tr>
		<tr>
			<td>Islet-1 antibody</td>
			<td>DSHB</td>
			<td>39.3F7</td>
			<td>1:100 dilution, RRID: AB_1157901</td>
		</tr>
		<tr>
			<td>Netwell plate</td>
			<td>Corning</td>
			<td>3478</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurofilament antibody</td>
			<td>Sigma-Aldrich</td>
			<td>N4142</td>
			<td>1:1000 dilution, RRID: AB_477272</td>
		</tr>
		<tr>
			<td>Parvalbumin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>P3088</td>
			<td>1:10000 dilution, RRID: AB_477329</td>
		</tr>
		<tr>
			<td>SNAP25 antibody</td>
			<td>Abcam</td>
			<td>ab66066</td>
			<td>1:1000 dilution, RRID: AB_2192052</td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe photoshop</td>
			<td>ADOBE</td>
			<td></td>
			<td>image editing software</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>LEICA</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Image-Pro Plus 7.0</td>
			<td>OlYMPUS</td>
			<td></td>
			<td>commercial image processing software package</td>
		</tr>
	</tbody>
</table>

dissecting cell-autonomous function of fragile x mental retardation protein in an auditory circuit by in ovo electroporation

脆性X染色体智力低下蛋白（FMRP）是一种调节局部蛋白质翻译的mRNA结合蛋白。FMRP丧失或功能障碍导致脆性X综合征（FXS）的神经元和突触活动异常，其特征是智力障碍，感觉异常和社会沟通问题。FMRP功能和FXS发病机制的研究主要在转基因动物中使用 Fmr1 （编码FMRP的基因）敲除进行。在这里，我们 报告了一种体内 方法，该方法用于使用鸡胚胎确定FMRP在电路组装和突触形成期间的细胞自主功能。该方法采用含有 Fmr1 小发夹RNA（shRNA）和EGFP报告基因的药物诱导载体系统的阶段，位点和方向特异性电穿孔。通过这种方法，我们在听觉神经节（AG）及其脑干靶标之一大细胞核（NM）中实现了选择性FMRP敲低，从而在AG-NM电路中提供了组件特异性操作。此外，转染的镶嵌模式允许动物内对照和邻近神经元/纤维比较，以提高数据分析的可靠性和灵敏度。诱导载体系统提供基因编辑开始的时间控制，以最大限度地减少累积的发育影响。这些策略的组合为剖析FMRP在突触和电路发育中的细胞自主功能提供了一种创新工具。

通过在不同部位和不同发育阶段进行 卵 电穿孔，我们在听觉外周或听觉脑干中实现了选择性FMRP敲低。
新墨西哥州的FMRP敲低 如前所述，设计针对鸡Fmr1的小发夹RNA（shRNA）并将其克隆到Tet-On载体系统中20。 卵内电穿孔的设置如图1A所示。将带有快速绿色着色的质粒DNA在HH12下以r5 / r6水平注射到神经管中...

Watch this Scientific Journal Video about Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation at JoVE.com

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

利用 卵 电穿孔，我们设计了一种选择性转染鸡胚胎中听觉内耳和耳蜗核的方法，以实现在电路组装的离散期间脆性X智力迟钝蛋白的细胞组特异性敲低。

通过产 卵电 穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to ...

我们的协议是一种体内方法，用于研究FMRP在开发过程中的细胞自主功能。这有助于了解脆性X综合征的细胞类型特异性病理生理学。该方法采用含有Fmr1小发夹RNA的药物诱导载体系统的阶段，位点和方向特异性电穿孔。通过这种方法，我们在听觉回路中实现了选择性FMRP敲低。该方法也可用于研究听觉回路或前庭系统中其他基因的功能。首先将卵子水平放置，将胚胎定位在卵子的顶部，以便于操作。在 38 摄氏度下孵育 46 至 48 小时，直到汉堡和汉密尔顿或 HH 阶段 12，用于神经管转染，或 54 至 56 小时，直到 HH 13 进行耳囊转染。从孵化器中取出鸡蛋，一次一打鸡蛋。将种蛋放在孵化器外超过一小时会引入发育变化并降低活力。用手电筒从鸡蛋底部投射光线。胚胎的位置在蛋壳上显示为一个黑暗区域。用铅笔在蛋壳上标记胚胎的位置。用含有75%乙醇的纱布擦拭鸡蛋，并用剪刀尖端在鸡蛋的尖头钻一个孔。然后，用 18 号针头注射器取出两毫升白蛋白。确保孔仅足够大以允许针头插入。用纱布擦去任何泄漏的白蛋白，并用透明胶带密封孔。用透明胶带覆盖鸡蛋的顶部，以铅笔标记的黑暗区域为中心，以尽量减少裂缝并防止在开窗过程中掉落的贝壳碎片。用含有75%乙醇的纱布擦拭所有手术工具，并用75%乙醇擦拭胶带覆盖的区域。用一把小剪刀在铅笔标记的圆周周围剪一个一到两平方厘米的窗口。将剪刀平放，以免损坏下面的胚胎。将带窗的鸡蛋放在具有10倍目镜和2倍变焦的立体显微镜下。用墨水溶液填充一毫升注射器，然后安装27号针头。用镊子将针头弯曲成45度角。在显微镜下，小心地从opaqua区域的边缘戳出，然后将针头插入胚胎下方。注入约50微升墨水，这些墨水将扩散到透明区域下方，以进行胚胎可视化。墨水将形成深色背景，以便清晰地可视化胚胎。使用移液器拉拔器将玻璃毛细管拉入移液器。在解剖显微镜下，用镊子小心地将毛细管针的尖端打开直径至10至20微米。将移液器存放在储物盒中直至使用。接下来，用注射器通过移液器末端的橡胶管施加负压，用0.5至1微升的质粒混合物填充毛细管移液器。将卵子放在显微镜下，使胚胎垂直定向，尾巴靠近您。用一只手或三轴机械手握住毛细管移液器，并将移液器的尖端驱动到菱形 5 至 6。在尾对头的方向。轻轻地将吸头穿过卵黄素膜并插入背侧神经管，然后稍微撤回移液器，使吸头在神经管腔内。通过施加气压注入质粒混合物，直到有色质粒完全扩散到菱形5至6中，并延伸到菱形3和菱形4中。检查注射是否成功，这是当蓝色质粒溶液快速扩散到神经管而不泄漏时实现的。当泄漏发生时，蓝色会迅速褪色。注射后，立即在神经管的两侧放置一个铂双极电极。使用电穿孔器提供两个 12 伏和 50 毫秒持续时间的脉冲。观察双极电极末端的气泡，负极端的气泡更多。检查电穿孔是否成功，这是在有色质粒混合物进入电极正极附近的神经管组织时实现的。电穿孔后，小心地取出双极电极。用一块预切成两英寸见方的透明薄膜覆盖蛋壳上的窗口，并喷洒 75% 乙醇。将鸡蛋放回孵化器中。清洁双极电极，在盐水中提供 10 到 20 个 12 伏脉冲和 50 毫秒持续时间，然后再进行下一个鸡蛋。在显微镜下，将卵子放在胚胎垂直且尾巴靠近您的方式放置。握住毛细管移液器，在背外侧方向轻轻戳右耳囊。用气压注入质粒混合物，直到耳囊充满蓝色溶液。检查注射是否成功，这是当蓝色质粒混合物被限制在耳囊内并且不泄漏时实现的。注射后立即将双极电极放在耳囊上，并将正侧和负侧分别放置在耳囊的前方和后方。使用电穿孔器提供两个 12 伏和 50 毫秒持续时间的脉冲。检查电穿孔是否成功，这是当蓝色质粒混合物进入电极正极附近的耳囊组织时实现的。电穿孔后，小心地取出双极电极，并用透明薄膜覆盖蛋壳上的窗口。最后，将鸡蛋放回孵化器。代表性图像显示了胚胎第二天或E2的神经管转染和人肉处理后胚胎第三天或E3的示例。此处显示了横截面上具有Fmr1 shRNA和EGFP的转染细胞。表达EGFP的细胞局限于神经管和脑干的一侧。此处显示了E15处的横截面，转染侧有表达EGFP的磁细胞核或NM神经元。在对侧，在海带核或NL的腹侧部分可以看到含EGFP的轴突。与邻近的非转染神经元相比，转染的NM神经元中的FMRP免疫反应性显着降低，验证了Fmr1 shRNA的敲低效果。此处显示了E19处伴FMRP免疫反应性的听神经节。听觉神经节神经元未转染。一些来源于颅神经嵴细胞的神经胶质细胞被转染。代表性图像显示耳囊转染后耳囊管中的FMRP敲低。E9的听觉导管表现出广泛的EGFP荧光。在横截面上，表达EGFP的细胞位于基底和听神经节中。与邻近的非转染毛细胞相比，转染毛细胞中的FMRP免疫反应性降低，验证了Fmr1 shRNA的敲低效果。同样，FMRP免疫反应性在转染的听觉神经节神经元中显着降低。转染的听觉神经节神经元的中心投影通过具有特征性终球末端的听觉神经追踪到脑干。在尝试此程序时，最重要的是控制电穿孔的位点和方向以进行选择性转染。按照此过程，可以进行单细胞类型填充，免疫染色，细胞分选，然后使用质谱或单细胞测序来揭示形态学和生化水平的变化。该技术还可以选择性地编辑其他基因，在干回路的听觉中具有时间控制和成分特异性，并且可以修改以操纵前庭系统。

Research

JoVE Journal

Neuroscience

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

TMS：使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

调查轻度创伤性脑损伤海马电路功能的改变

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

In ovo Expression of MicroRNA in Ventral Chick Midbrain

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

通过产 卵电 穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能