We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission&#8217;s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the &#8220;Stem Cells Call for Proposals.&#8221; This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by &#8220;Ente Cassa di Risparmio di Firenze&#8221;.

1，轴突标签<ol><li>攀登纤维可以通过注入缀合到高分子量的葡聚糖或质粒/病毒诱导荧光蛋白26-29的表达任一有机染料进行标记。在这个协议中，该有机染料的Alexa Fluor右旋糖酐488被注入到下橄榄标记攀爬纤维和可视化它们在小脑皮质（ 图1）。这里描述的所有程序已批准由卫生意大利外交部。 </li><li>通过在微量拉马拉它准备在玻璃毛细管。修剪的玻璃毛细管的尖端用剪刀直到外部直径大约是30微米。 </li><li>加载毛细管用1-2微升的葡聚糖 - 缀合的染料。 </li><li>在开始之前，灭菌在高压釜中所有的手术器械。在实验过程中，通过使用玻璃珠灭菌降低污染风险。 </li><li>麻醉小鼠（9-12周）通过腹膜内注射氯胺酮（90毫克/千克）和赛拉嗪（10毫克/千克）的。确保动物用尾巴和/或脚趾捏完全镇静。使用上的眼睛兽医药膏，以防止肌肤干燥，同时在麻醉下。 </li><li>刮颈部以上的头发和一台刀片或脱毛霜。用优碘，交替用70％乙醇擦拭手术部位的两倍。 </li><li>把鼠标放在一个立体持有人。保持动物温暖，加热垫。 </li><li>轻轻推动骨earbars旁边的耳朵，头部被牢牢地举行。旋转头以形成一个140°的角度与机身。所需的方向可​​以通过缓慢地变化的立体定向架的鼻架杆的高度来获得。 </li><li>应用利多卡因几滴在两耳之间的皮肤。使用镊子或刀片上的枕部下面的皮肤进行约1厘米的垂直切口。 </li><li>轻轻地由钝钳分离皮下组织和肌肉S中的解剖显微镜下。下推水平肌束并保持分开的两个垂直的肌肉分册，以暴露硬脑膜在枕骨大孔。 </li><li>使用格外小心，解剖用注射器针头或非常锋利的钳暴露脑干硬脑膜。少量的脑脊髓液可能会洒出来的硬脑膜已被切断时。 </li><li>在立体定向显微操作，旋转从垂直毛细管45°的固定器。将毛细管在中线，在小脑皮层的尾缘和第一颈椎之间的中点。插入吸移管以2mm​​距表面的深度。 </li><li>提供染料的0.5-1.5微升量超过10-15分钟。显微注射点胶系统允许在毛细管中的液体控制压力输送（脉冲压力= 20磅，脉冲持续时间为3-4毫秒，频率脉冲传递= 12赫兹）。 </li><li>离开毛细管到位15分钟，然后slowlY（2-3分钟），将其删除。微妙地重新对准肌肉和缝合上面的皮肤。继续通过应用一些盐水在整个过程以及水合肌肉。 </li><li>以避免脱水，注入皮下0.5毫升0.9％的NaCl。保持动物在笼子里加热，直到从麻醉中完全恢复。 </li><li>每日皮下注射给药卡洛芬（5毫克/公斤）的手术后2-3天。 </li></ol> 2，对小脑皮质光学窗口注：染料是从下橄榄核向上运送2周至小脑皮层的CFs的端子（参见图1）。人烟稀少标记的CF可以，可以如下进行永久性颅窗下进行可视化： <ol><li>消毒所有的手术器械。 </li><li>麻醉如上所述鼠标。用脚趾捏来检查动物是完全镇静。 </li><li>剃了头发的脑袋上，抹了SK在用70％酒精和优碘的交替刷卡和应用眼膏。鼠标放置在一个立体框架和定位，以耳棒牢牢握住头。 </li><li>皮下给药地塞米松（0.2毫克/千克）和卡洛芬（5毫克/公斤），以防止脑和可能的炎症的肿胀在颅窗口部位。 </li><li>应用利多卡因对颅骨上方的皮肤下降，切瓣，从两耳之间上面的眼睛。用手术刀刮之前重新申请利多卡因溶液到骨膜。 </li><li>使用牙钻颅骨的小脑上面薄薄的半圆形区域。窗口通常置于中央和朝后缝合，从而使颅骨的深度是在整个半圆的周长几乎恒定。定位所述窗口太近的λ缝合和重复钻孔在这个很厚区域可能导致水肿的脑和地层的过热。 </li><li>应用止血药海绵上，以防出血的头骨。 </li><li>切玻璃罩成两半用剃刀刀片和镊子的帮助。检查玻璃罩比骨岛较大，并且具有去除骨用镊子的前瓣形状相似;如果不是，重塑玻璃罩或者选择另外一个。请勿触摸硬膜用镊子同时绘制脱骨。在硬脑膜仔细放下盖玻片。注意：在硬膜玻璃的整个表面上的粘附被预言的持久颅窗口。 </li><li>通过反复冲洗用生理盐水溶液，用棉枝干燥玻璃下去除气泡。这也将有助于消除一些残留的血液可能会从细小的毛细血管溢出定位后的玻璃。用亚克力胶水和牙科水泥30的混合物密封窗。 </li><li>保持动物加热垫在恢复室中，直到从麻醉中完全恢复。不要再把老鼠的笼子，直到完全恢复;不要随意放置，直到他们恢复了足够的意识，以保持胸骨斜卧。 </li><li>每日皮下注射给药卡洛芬（5毫克/公斤）的手术后2-3天。 </li></ol> 3， 在体内多光子激光干切断<ol><li>等待2-7天，手术进行的第成像会议之前。内置这里使用双光子显微镜自定义设置有钛：蓝宝石激光光源（120飞秒的脉冲宽度，90 MHz的重复频率），这是第一个由一对检流镜的扫描，然后通过一个水浸泡20X聚焦到试样物镜（NA 0.95，WD 2mm）的。一种闭环压电阶段进行轴向位移到400的目标微米。光电倍增管收集荧光信号。 </li><li>麻醉动物，然后放置在头部立体定向架，使颅窗口奠定平行objecti已经焦平面。 </li><li>探索与TPF成像皮质颅窗下。选择要损坏之中最亮的那些轴突。 </li><li>获得的Z堆叠（通常视场为100×100微米2，在512×512象素，2微米z轴步骤）以获得3D-重建目标轴突。在成像的容积，选择病灶部位上的轴突分支，主要在于平行于焦平面。这个方向可以快速得分的形态变化，这是预测一个成功的夹层。 </li><li>照射具有高能量剂量在CF的远端部分所选择的点。这是通过Labview的软件来完成：增加激光功率5至10倍比用于成像（≈300毫瓦，物镜后测得）的功率多，设置的检流计反射镜的位置做线扫描（〜4 - 10μm）的病变部位，打开激光快门为400 - 600毫秒。定向线扫描垂直于平面的CF完全横切轴突。照射明亮的斑点（ 如 。，膨体）通常有助于解剖轴突。该波长的激光干切断是用于成像（920 nm）的相同。 </li><li>通常在照射区域中的轴突的荧光瞬时减小由于光漂白。获得一个堆栈的相同区域几分钟后照射，以确保在消融得到了很好的执行。如果该区域开始肿胀和轴突形成的珠状结构，神经元已被成功解剖。在这种情况下，在几小时内（从几十分钟至24小时）的轴突的远端部分会变质而完全消失。 </li><li>不幸的是要产生一个完整的干切断能量剂量并不总是可以预测的。如果照射的唯一结果是光漂白，荧光应当迅速恢复由于染料扩散，照射应再次重复，以有效LY解剖轴突。另外，在壳体膨胀的迹象并不内出现5 - 20分钟，或肿胀后跟恢复（类似于已经描述用于激光dendrotomy，看到的Allegra卡罗等人 23），用新的扫描尝试再次增加停留时间和/或激光器功率。 </li><li>监视轴突的最终重构的跟随激光干切断与双光子的体内成像的天（ 图2和3）。血管图案被用作参考的模式来检索解剖轴突在随后的日子里。 </li><li>皮下管理卡洛芬（5毫克/公斤）为实验的整个持续时间每个成像会议。 </li></ol>

<ol>
	<li>Strata, P., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+olivocerebellar+pathway.">Plasticity of the olivocerebellar pathway.</a> Trends Neurosci. 21, 407-413 (1998).</li><li>Carulli, D., Buffo, A., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reparative+mechanisms+in+the+cerebellar+cortex.">Reparative mechanisms in the cerebellar cortex.</a> Prog Neurobiol. 72, 373-398 (2004).</li><li>Zipfel, W., Williams, R., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nature Biotechnology. 21, 1369-1377 (2003).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2, 932-940 (2005).</li><li>Sacconi, 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=In+vivo+multiphoton+nanosurgery+on+cortical+neurons.">In vivo multiphoton nanosurgery on cortical neurons.</a> J Biomed Opt. 12, 050502 (2007).</li><li>Allegra Mascaro, A. 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=In+vivo+single+branch+axotomy+induces+GAP-43-dependent+sprouting+and+synaptic+remodeling+in+cerebellar+cortex.">In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex.</a> Proc Natl Acad Sci USA. 110, 10824-10829 (2013).</li><li>Ylera, B., 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=Chronically+CNS-injured+adult+sensory+neurons+gain+regenerative+competence+upon+a+lesion+of+their+peripheral+axon.">Chronically CNS-injured adult sensory neurons gain regenerative competence upon a lesion of their peripheral axon.</a> Curr Biol. 19, 930-936 (2009).</li><li>Canty, A. J., 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=In-vivo+single+neuron+axotomy+triggers+axon+regeneration+to+restore+synaptic+density+in+specific+cortical+circuits.">In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits.</a> Nat Commun. 4, 2038 (2013).</li><li>Canty, A. J., 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=Synaptic+elimination+and+protection+after+minimal+injury+depend+on+cell+type+and+their+prelesion+structural+dynamics+in+the+adult+cerebral+cortex.">Synaptic elimination and protection after minimal injury depend on cell type and their prelesion structural dynamics in the adult cerebral cortex.</a> J Neurosci. 33, 10374-10383 (2013).</li><li>Waller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+Section+of+the+Glossopharyngeal+and+Hypoglossal+Nerves+of+the+Frog,+and+Observations+of+the+Alterations+Produced+Thereby+in+the+Structure+of+Their+Primitive+Fibres.">Experiments on the Section of the Glossopharyngeal and Hypoglossal Nerves of the Frog, and Observations of the Alterations Produced Thereby in the Structure of Their Primitive Fibres.</a> Philosophical Transactions of the Royal Society of London. 140, 423-429 .</li><li>Hilliard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+degeneration+and+regeneration+a+mechanistic+tug+of+war.">Axonal degeneration and regeneration a mechanistic tug of war.</a> J Neurochem. 108, 23-32 (2009).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, 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+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">In vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat Med. 11, 572-577 (2005).</li><li>Luo, L., O'Leary, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+retraction+and+degeneration+in+development+and+disease.">Axon retraction and degeneration in development and disease.</a> Annu Rev Neurosci. 28, 127-156 (2005).</li><li>Chauvet, N., Prieto, M., Alonso, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tanycytes+present+in+the+adult+rat+mediobasal+hypothalamus+support+the+regeneration+of+monoaminergic+axons.">Tanycytes present in the adult rat mediobasal hypothalamus support the regeneration of monoaminergic axons.</a> Exp Neurol. 151, 1-13 (1998).</li><li>Morrison, E. E., Costanzo, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+olfactory+sensory+neurons+and+reconnection+in+the+aging+hamster+central+nervous+system.">Regeneration of olfactory sensory neurons and reconnection in the aging hamster central nervous system.</a> Neurosci Lett. 198, 213-217 (1995).</li><li>Cafferty, W. B., McGee, A. W., Strittmatter, S. M. <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+therapeutics+regeneration+or+sprouting+or+plasticity.">Axonal growth therapeutics regeneration or sprouting or plasticity.</a> Trends Neurosci. 31, 215-220 (2008).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Two-Photon+Excitation+Microscopy+and+Its+Applications+to+Neuroscience.">Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Feng, G., 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Tomomura, M., Rice, D. S., Morgan, J. I., Yuzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Purkinje+cells+by+fluorescence-activated+cell+sorting+from+transgenic+mice+that+express+green+fluorescent+protein.">Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein.</a> Eur J Neurosci. 14, 57-63 (2001).</li><li>De Paola, V., Arber, S., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptors+regulate+dynamic+equilibrium+of+presynaptic+terminals+in+mature+hippocampal+networks.">AMPA receptors regulate dynamic equilibrium of presynaptic terminals in mature hippocampal networks.</a> Nat Neurosci. 6, 491-500 (2003).</li><li>Holtmaat, A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+structural+synaptic+plasticity+in+the+mammalian+brain.">Experience-dependent structural synaptic plasticity in the mammalian brain.</a> Nature reviews. Neuroscience. 10, 647-658 (2009).</li><li>De Paola, V., 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=Cell+type-specific+structural+plasticity+of+axonal+branches+and+boutons+in+the+adult+neocortex.">Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex.</a> Neuron. 49, 861-875 (2006).</li><li>Allegra Mascaro, A., Sacconi, L., Pavone, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+nanosurgery+in+live+brain.">Multi-photon nanosurgery in live brain.</a> Frontiers in neuroenergetics. 2, 21 (2010).</li><li>Rossi, F., van der Want, J., Wiklund, L., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinnervation+of+cerebellar+Purkinje+cells+by+climbing+fibres+surviving+a+subtotal+lesion+of+the+inferior+olive+in+the+adult+rat.+II.+Synaptic+organization+on+reinnervated+Purkinje+cells.">Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. II. Synaptic organization on reinnervated Purkinje cells.</a> The Journal of comparative neurology. 308, 536-554 (1991).</li><li>Carulli, D., Buffo, A., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reparative+mechanisms+in+the+cerebellar+cortex.">Reparative mechanisms in the cerebellar cortex.</a> Progress in neurobiology. 72, 373-398 (2004).</li><li>Grasselli, G., Mandolesi, G., Strata, P., Cesare, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+sprouting+and+axonal+atrophy+in+cerebellar+climbing+fibres+following+in+vivo+silencing+of+the+growth-associated+protein+GAP-43.">Impaired sprouting and axonal atrophy in cerebellar climbing fibres following in vivo silencing of the growth-associated protein GAP-43.</a> PLoS One. 6, e20791 (2011).</li><li>Nishiyama, H., Fukaya, M., Watanabe, M., Linden, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+motility+and+its+modulation+by+activity+are+branch-type+specific+in+the+intact+adult+cerebellum.">Axonal motility and its modulation by activity are branch-type specific in the intact adult cerebellum.</a> Neuron. 56, 472-487 (2007).</li><li>Shinoda, Y., Sugihara, I., Wu, H. S., Sugiuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entire+trajectory+of+single+climbing+and+mossy+fibers+in+the+cerebellar+nuclei+and+cortex.">The entire trajectory of single climbing and mossy fibers in the cerebellar nuclei and cortex.</a> Prog Brain Res. 124, 173-186 (2000).</li><li>Strata, P., Tempia, F., Zagrebelsky, M., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+trophic+interactions+between+climbing+fibres+and+Purkinje+cells+in+the+rat+cerebellum.">Reciprocal trophic interactions between climbing fibres and Purkinje cells in the rat cerebellum.</a> Prog Brain Res. 114, 263-282 (1997).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. (12), (2008).</li></ol>

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这个协议说明如何来标记下橄榄的神经元用荧光染料。随后，为了在小脑皮层进行颅窗口的方法进行说明。这种技术提供的光纤接入到olivocerebellar神经元，攀登纤维的端部。不幸的是，这两个标签和开颅手术的结果是相当低的，即使在熟练的操作人员的手（通常为1出3只小鼠的标记，和1出3颅窗1-2周后依然清晰）。 在活小鼠进行激光干切断的程序，然后提交。这种方法允许在?...

从机械性损伤，有毒侮辱或者神经退行性疾病导致轴突切断术之后通常是从细胞体分离10-13轴突的远端部分变性。除了 ​​少数例外2,7,14,15，在成年动物的中枢神经系统轴突切断通常无法激活再生程序16。 鲜为人知的是，在细胞和亚细胞水平退化性事件的实时动态。的新策略，用于限制神经元损伤，促进神经元的再生的发展需要，作为第一步，阐明由奇受伤的神经细胞退化和再生的机制。本研究是最直接处理通过监测体内的单个神经元的动态。而单光子荧光成像技术是将可见光的强烈散射的限制，双光子激发到达里深皮质层已经小鼠的亚细胞分辨率3,4,17。利用转基因小鼠中，荧光蛋白选择性地表达于神经元18-20的亚群，TPF显微镜已经发展在体内 21,22中应用到突触可塑性和轴突伸长的探索。奇受损神经元的T到他的能力再生后的损伤可通过耦合在体内监测通过双光子成像损伤的专门针对感兴趣的轴突的模型研究。飞秒激光脉冲的多光子吸收被用于破坏单 ​​个树突甚至单棘5,23。此外，这种损伤模式允许单个切割轴突分支，而不会中断接触枝晶6。在解剖，允许特定的神经元群的特征再生轴突一旦受损，小脑登山纤维（CFS）的情况下是一个有用的模型SINCE他们保留显着的塑料性能，即使在成年动物24,25损伤后。最近，CF的长期成像表明，这些轴突能够再生长在遵循激光干切断6的日子。 本协议描述了如何通过顺行示踪标记olivocerebellar神经元及其轴突的伸长率。一旦所感兴趣的神经元被荧光标记，它们可以被重复地用于下一个窗口颅周或数月的任意时间点进行监测。由激光干切断体内解剖单个轴突分枝的要领进行说明。 这里介绍的技术提供了新的见解体内轴突重塑的机制，可能有助于治疗策略的开发，以限制神经元变性和促进轴突再生。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument Inc</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0720</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser, 120 fs width pulses,&#160; 90 MHz repetition rate</td>
			<td>Coherent</td>
			<td>Chameleon&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spongostan, haemostatic sponge&#160;</td>
			<td>Ferrosan</td>
			<td>MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometric mirrors&#160;</td>
			<td>GSI Lumonics</td>
			<td>VM500+</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier modules&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>H7710-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW System Design Software</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Voren, 1 mg/ml (dexamethasone-21- isonicotinate)</td>
			<td>Boheringer Ingelheim</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rymadil (carprofen)&#160;</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine clorohydrate 2%</td>
			<td>ATI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 dextran</td>
			<td>Life Technologies</td>
			<td>D22910</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser nanosurgery of cerebellar axons in vivo

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 understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

该协议描述了如何在单个神经元的轴突进行标记， 体内成像和激光干切断。实验的时间线示于图1。 的CF标记的Alexa Fluor 488葡聚糖和颅窗口通过体内双光子显微镜下观察的例子报道于图2。如先前报道6,27，上行分支在整个数天的观察期显示高稳定性。 图3显示了轴突切断由CF的单个分支的激光干切...

Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

Laser Nanosurgery of Cerebellar Axons In Vivo

双光子成像，其耦合到激光nanodissection，是有用的工具来研究退化和再生过程中对中枢神经系统具有亚细胞分辨率。该协议显示了如何标记，图像，并剖析单个登山纤维在体内小脑皮质。

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 ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

Research

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

8.1K 次观看.  University of Florence. 双光子成像，其耦合到激光nanodissection，是有用的工具来研究退化和再生过程中对中枢神经系统具有亚细胞分辨率。该协议显示了如何标记，图像，并剖析单个登山纤维在体内小脑皮质。 