Name: methodology for biomimetic chemical neuromodulation of rat retinas with the neurotransmitter glutamate in vitro
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Description: Watch this Scientific Journal Video about Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro at JoVE.com

论文中介绍的工作得到了国家科学基金会的支持, 研究和创新领域的新兴前沿 (NSF-EFRI) 方案赠款0938072。本文的内容完全是作者的责任, 不一定代表 NSF 的官方观点。作者还希望感谢 Dr. Samsoon Inayat 为他的工作设计和测试的初步实验设置的化学刺激和 Mr. 温 Raghunathan 为他的工作设计, 制作, 并评价了多端口微流控装置使用这项研究。

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	<li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. , (2011).</li><li>Fritsche, L. G., Fariss, R. N., Stambolian, D., Abecasis, G. R., Curcio, C. A., Swaroop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Macular+Degeneration:+Genetics+and+Biology+Coming+Together.">Age-Related Macular Degeneration: Genetics and Biology Coming Together.</a> Annu Rev Genomics Hum Genet. 15, 151-171 (2014).</li><li>Marc, R. 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=Neural+reprogramming+in+retinal+degeneration.">Neural reprogramming in retinal degeneration.</a> Invest Ophthalmol Vis Sci. 48, 3364-3371 (2007).</li><li>Jones, B. W., Kondo, M., Terasaki, H., Lin, Y., McCall, M., Marc, 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=Retinal+remodeling.">Retinal remodeling.</a> Jpn J Ophthalmol. 56, 289-306 (2012).</li><li>Soto, F., Kerschensteiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+remodeling+of+neuronal+circuits+in+early+retinal+degeneration.">Synaptic remodeling of neuronal circuits in early retinal degeneration.</a> Front Cell Neurosci. 9, (2015).</li><li>Trenholm, S., Awatramani, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+spontaneous+activity+in+the+degenerating+retina.">Origins of spontaneous activity in the degenerating retina.</a> Front Cell Neurosci. 9, (2015).</li><li>Euler, T., Schubert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Independent+Oscillatory+Networks+in+the+Degenerating+Retina.">Multiple Independent Oscillatory Networks in the Degenerating Retina.</a> Front Cell Neurosci. 9, (2015).</li><li>Boye, S. E., Boye, S. L., Lewin, A. S., Hauswirth, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+Review+of+Retinal+Gene+Therapy.">A Comprehensive Review of Retinal Gene Therapy.</a> Mol Ther. 21, 509-519 (2013).</li><li>Schwartz, S. 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=Human+embryonic+stem+cell-derived+retinal+pigment+epithelium+in+patients+with+age-related+macular+degeneration+and+Stargardt's+macular+dystrophy:+follow-up+of+two+open-label+phase+1/2+studies.">Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies.</a> The Lancet. 385, 509-516 (2015).</li><li>Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+Transplantation+in+Late+Stage+Retinal+Degeneration.">Photoreceptor Transplantation in Late Stage Retinal Degeneration.</a> Invest Ophthalmol Vis Sci. 57, (2016).</li><li>Zrenner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fighting+blindness+with+microelectronics.">Fighting blindness with microelectronics.</a> Sci Transl Med. 5, (2013).</li><li>Humayun, M. S., de Juan, E., Dagnelie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bionic+Eye:+A+Quarter+Century+of+Retinal+Prosthesis+Research+and+Development.">The Bionic Eye: A Quarter Century of Retinal Prosthesis Research and Development.</a> Ophthalmol. 123, S89-S97 (2016).</li><li>Cruz, 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=The+Argus+II+epiretinal+prosthesis+system+allows+letter+and+word+reading+and+long-term+function+in+patients+with+profound+vision+loss.">The Argus II epiretinal prosthesis system allows letter and word reading and long-term function in patients with profound vision loss.</a> Br J Ophthalmol. 97, 632-636 (2013).</li><li>Zrenner, 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=Subretinal+electronic+chips+allow+blind+patients+to+read+letters+and+combine+them+to+words.">Subretinal electronic chips allow blind patients to read letters and combine them to words.</a> P R Soc B. 278, 1489-1497 (2011).</li><li>Stronks, H. C., Dagnelie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+performance+of+the+Argus+II+retinal+prosthesis.">The functional performance of the Argus II retinal prosthesis.</a> Expert Rev Med Devices. 11, 23-30 (2014).</li><li>Stingl, K., 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=Artificial+vision+with+wirelessly+powered+subretinal+electronic+implant+alpha-IMS.">Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS.</a> P R Soc B. 280, (2013).</li><li>Rizzo, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+retinal+prosthetic+research:+the+Boston+Retinal+Implant+Project.">Update on retinal prosthetic research: the Boston Retinal Implant Project.</a> J Neuroophthalmol. 31, 160-168 (2011).</li><li>Ayton, L. 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=First-in-Human+Trial+of+a+Novel+Suprachoroidal+Retinal+Prosthesis.">First-in-Human Trial of a Novel Suprachoroidal Retinal Prosthesis.</a> PLoS ONE. 9, e115239 (2014).</li><li>Chuang, A. T., Margo, C. E., Greenberg, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+implants:+a+systematic+review.">Retinal implants: a systematic review.</a> Br J Ophthalmol. 98, 852-856 (2014).</li><li>Cai, C., Twyford, P., Fried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+retinal+neurons+to+high-frequency+stimulation.">The response of retinal neurons to high-frequency stimulation.</a> J Neural Eng. 10, 036009 (2013).</li><li>Eiber, C. D., Lovell, N. H., Suaning, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attaining+higher+resolution+visual+prosthetics:+a+review+of+the+factors+and+limitations.">Attaining higher resolution visual prosthetics: a review of the factors and limitations.</a> J Neural Eng. 10, 011002 (2013).</li><li>Humayun, M., Propst, R., de Juan, E., McCormick, K., Hickingbotham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bipolar+surface+electrical+stimulation+of+the+vertebrate+retina.">Bipolar surface electrical stimulation of the vertebrate retina.</a> Arch Ophthalmol. 112, 110-116 (1994).</li><li>Zrenner, 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=Can+subretinal+microphotodiodes+successfully+replace+degenerated+photoreceptors?.">Can subretinal microphotodiodes successfully replace degenerated photoreceptors?.</a> Vision Res. 39, 2555-2567 (1999).</li><li>Majji, A. B., Humayun, M. S., Weiland, J. D., Suzuki, S., D'Anna, S. A., de Juan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Histological+and+Electrophysiological+Results+of+an+Inactive+Epiretinal+Electrode+Array+Implantation+in+Dogs.">Long-Term Histological and Electrophysiological Results of an Inactive Epiretinal Electrode Array Implantation in Dogs.</a> Invest Ophthalmol Vis Sci. 40, 2073-2081 (1999).</li><li>Peterman, M. C., Noolandi, J., Blumenkranz, M. S., Fishman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+chemical+release+from+an+artificial+synapse+chip.">Localized chemical release from an artificial synapse chip.</a> PNAS. 101, 9951-9954 (2004).</li><li>Finlayson, P. G., Iezzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+stimulation+of+retinal+ganglion+cells+in+normal+and+s334ter-4+rat+retinas:+a+candidate+for+a+neurotransmitter-based+retinal+prosthesis.">Glutamate stimulation of retinal ganglion cells in normal and s334ter-4 rat retinas: a candidate for a neurotransmitter-based retinal prosthesis.</a> Invest Ophthalmol Vis Sci. 51, 3619-3628 (2010).</li><li>Inayat, S., Rountree, C. M., Troy, J. B., Saggere, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+stimulation+of+rat+retinal+neurons:+feasibility+of+an+epiretinal+neurotransmitter-based+prosthesis.">Chemical stimulation of rat retinal neurons: feasibility of an epiretinal neurotransmitter-based prosthesis.</a> J Neural Eng. 12, 016010 (2015).</li><li>Rountree, C. M., Inayat, S., Troy, J. B., Saggere, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+stimulation+of+the+retina+with+subretinally+injected+exogenous+neurotransmitter:+A+biomimetic+alternative+to+electrical+stimulation.">Differential stimulation of the retina with subretinally injected exogenous neurotransmitter: A biomimetic alternative to electrical stimulation.</a> Sci Rep. 6, 38505 (2016).</li><li>Ray, A., Sun, G. J., Chan, L., Grzywacz, N. M., Weiland, J., Lee, E. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+alterations+in+retinal+neurons+in+the+S334ter-line3+transgenic+rat.">Morphological alterations in retinal neurons in the S334ter-line3 transgenic rat.</a> Cell Tissue Res. 339, 481-491 (2010).</li><li>Martinez-Navarrete, G., Seiler, M. J., Aramant, R. B., Fernandez-Sanchez, L., Pinilla, I., Cuenca, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+two+lines+of+transgenic+S334ter+rats.">Retinal degeneration in two lines of transgenic S334ter rats.</a> Exp Eye Res. 92, 227-237 (2011).</li><li>. Sigma Aldrich Ames Medium Product Information Sheet Available from: <a target="_blank" href="https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/a1420pis.pdf">https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/a1420pis.pdf</a> (2017)</li><li>Reinhard, K., 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=Step-By-Step+instructions+for+retina+recordings+with+perforated+multi+electrode+arrays.">Step-By-Step instructions for retina recordings with perforated multi electrode arrays.</a> PLoS ONE. 9, e106148 (2014).</li><li>Izumi, Y., Kirby, C. O., Benz, A. M., Olney, J. W., Zorumski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+cell+swelling,+glutamate+uptake,+and+excitotoxic+neurodegeneration+in+the+isolated+rat+retina.">M&#252;ller cell swelling, glutamate uptake, and excitotoxic neurodegeneration in the isolated rat retina.</a> Glia. 25, 379-389 (1999).</li><li>Tunnicliff, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+uptake+by+chick+retina.">Glutamate uptake by chick retina.</a> Biochem J. 150, 297-299 (1975).</li><li>Schwartz, E. A., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+of+glutamate+and+sodium+co-transport+in+a+glial+cell+of+the+salamander+retina.">Electrophysiology of glutamate and sodium co-transport in a glial cell of the salamander retina.</a> J Physiol (Lond). 426, 43-80 (1990).</li><li>Muller, A., Maurin, L., Bonne, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+glutamate+uptake+in+the+retina.">Free radicals and glutamate uptake in the retina.</a> Gen Pharmacol- Vasc S. 30, 315-318 (1998).</li><li>Dhingra, N. K., Kao, Y. -. H., Sterling, P., Smith, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+threshold+of+a+brisk-transient+ganglion+cell+in+vitro.">Contrast threshold of a brisk-transient ganglion cell in vitro.</a> J of Neurophysiol. 89, 2360-2369 (2003).</li><li>Ahlers, M. T., Ammerm&#252;ller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+precise+temperature+control+of+isolated+nervous+tissue+under+optical+access:+Application+to+multi-electrode+recordings.">A system for precise temperature control of isolated nervous tissue under optical access: Application to multi-electrode recordings.</a> J of Neurosci Methods. 219, 83-91 (2013).</li><li>Feke, G. T., Tagawa, H., Deupree, D. M., Goger, D. G., Sebag, J., Weiter, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+in+the+normal+human+retina.">Blood flow in the normal human retina.</a> Invest Ophthalmol Vis Sci. 30, 58-65 (1989).</li><li>Purves, 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=The+Retina.">The Retina.</a> Neuroscience. , (2001).</li></ol>

作者没有什么可透露的。

这里介绍的方法展示了一个独特的神经刺激模式, 其中视网膜神经元的化学刺激, 注射当地神经递质化学物质进入视网膜的地下体外。这种化学刺激技术在传统的电刺激技术中提供了一些好处, 包括选择性和靶神经元的高聚焦特异性。上面的协议详细说明了如何小体积的神经递质谷氨酸注射在目标视网膜神经元附近使用单玻璃微或自定义微流控流体设备引出在生理学上重要的研资反应。虽然?...

感光性退行性疾病, 如视网膜色素变性和年龄相关的黄斑变性, 是导致视力丧失的可继承原因, 目前是无法治愈的1,2。尽管这些疾病是由各种特定的基因突变引起的, 但感光变性疾病是由视网膜感光细胞的逐渐丧失而引起的, 最终导致失明。光的丢失引发了整个视网膜的广泛重塑, 但存活的视网膜神经元, 包括双极细胞和视网膜, 仍然完好无损, 即使在感光变性的高级阶段也相对正常,3 ,4,5,6,7。
这些疾病的机制和病理特点已经很好的特征3,4,5,6,7 , 但有效的治疗仍然难以捉摸。在过去的三年中, 世界各地的研究人员已经研究了多种治疗方法, 以恢复视力的那些受影响的感光变性疾病, 包括基因治疗8, 干细胞治疗9,视网膜移植10, 人工刺激11,12存活的视网膜神经元。其中, 最有临床意义的是视网膜假体, 这是人工神经装置, 传统上使用了一系列电极, 以电刺激双极细胞或视网膜在特定模式的目标在患者中创建人工视觉感知11。当前世代电子假肢, 例如守卫 II13和阿尔法 IMS14设备, 获得了临床批准, 并且初步研究表明, 他们能改善患者的生活质量通过恢复测量视觉使用两个前 (视网膜的前面) 和视网膜 (视网膜的后面) 植入的设备15,16。世界各地的研究小组正在努力推进视网膜假肢超越这些 first-generation 设备的成功17,18,19,20 ,但已面临困难设计一个电子假肢能够恢复高视力视力低于法律失明水平的病人。最近的研究表明, 实现更高的空间分辨率比目前的代为主假肢是挑战, 因为电荷注入限制, 这就需要使用大的电极来安全地刺激视网膜神经元以空间分辨率为代价,即视觉灵敏度11,21。此外, 电刺激是进一步有限的, 因为它通常刺激所有附近的细胞, 从而引起病人的不自然和混淆的看法, 很大程度上是因为它是一个固有的非自然刺激范例21。然而, 早期的电刺激成功表明, 人工神经可以是一种有效的治疗感光变性疾病。这导致一个假设, 更有效的治疗可能是可以通过刺激视网膜与神经递质化学品, 在化学突触沟通的自然代理人。该方法的目的是探讨化学刺激的治疗可行性, 旨在模拟视网膜神经元之间的突触通信的自然系统, 作为电刺激的仿生替代品。用于视网膜假体
对化学性视网膜假体的治疗性化学刺激的概念的翻译依赖于化学活化的目标视网膜神经元, 少量的本地神经递质, 如谷氨酸, 通过微流控物释放一种装置, 包括大量的 microports 以响应视觉刺激。这样, 一个化学的视网膜假体将本质上是一个仿生的人工感光层, 把光子自然地到达视网膜的化学信号。由于这些化学信号在正常的视网膜信号中使用相同的神经递质, 并通过正常视觉通路所使用的相同突触通路刺激退化视网膜的存活的视网膜神经元, 由此产生的视觉通过一个化学的视网膜假体的知觉可以更自然和可理解相比, 通过一个电动假肢诱发。此外, 由于神经递质被释放的 microports 可以非常小, 并排列在高密度, 不像电极, 一个潜在的化学假体可能能够获得更多的焦点刺激和更高的空间分辨率比电子假肢。因此, 基于这些潜在的优势, 化学性视网膜假体提供了一个非常有前途的替代电气假肢。
然而, 直到最近, 对视网膜的化学刺激也相对较少。而电刺激的视网膜已经很好地表征了几十年的工作通过体外22,23,在体内23,24, 和临床研究13 ,14, 对化学刺激的研究仅限于少数体外工作25,26,27,28。耶齐和芬利森26和 Inayat et al.27分别使用单电极和多阵列 (前) 对视网膜的化学刺激进行了实验, 以记录视网膜神经元的谷氨酸诱发反应。最近, 朗特里et al.28显示了视网膜侧的谷氨酸和视网膜通路的差异刺激, 并记录了视网膜上多个部位的神经元反应。虽然这些工作已经初步确定了化学刺激的可行性, 但进一步的研究对于研究这种方法的许多方面是非常必要的, 除了这些方面, 到目前为止25,26,27,28, 并 fine-tune 的治疗刺激参数在两个体外和在体内动物模型之前, 把这个概念转化为化学视网膜假体如上所述。然而, 目前还没有确定的方法, 以完成化学刺激的视网膜在文献中使用的方法, 在以前的作品没有被描述的细节, 这将是必要的复制研究。因此, 本方法的基本原理是提供一个明确的框架来进行体外对视网膜的化学刺激, 对于那些有兴趣复制我们以前的研究的调查者27, 28或进一步推进这一新生的化学神经概念。
在这里, 我们演示了一种进行体外的方法, 对野生型大鼠 wholemount 视网膜神经元的化学刺激及感光变性大鼠模型的模拟疾病在人。在体外模型中开发这种刺激方法的基本原理是评估各种刺激参数的治疗范围, 并研究在中不可能或难以观察到的神经反应特性. 体内的模型, 特别是在最初的研究集中在评估这种方法的可行性。在这个过程中, 我们显示站点和同时多站点的视网膜的化学刺激通过提供少量的1毫米谷氨酸近靶视网膜神经元通过商业可用的单玻璃 micropipettes 和自定义多口微流控装置, 分别。虽然站点和多站点刺激完成了研究化学调节的治疗可行性的基本目标, 但它们都具有独特的优势。站点刺激, 这可能是完成与商业可用的 pre-pulled 玻璃 micropipettes, 可用于直接注入化学品的视网膜下在一个单一的地点, 并有助于调查, 如果可见的研资局穗率类似于视觉诱发光反应的反应可以在注射部位局部。另一方面, 多站点刺激, 需要专门制作的多端口微流控装置, 可以被用来在多个地点的视网膜表面上注入化学物质, 并有助于研究谷氨酸诱发 RCG反应模式对应于模式刺激研究中的谷氨酸注射模式。

<table><tbody><tr><td>Microelectrode array, perforated layout</td><td>Multi Channel Systems, GmbH</td><td>60pMEA200/30iR-Ti-pr</td><td>http://www.multichannelsystems.com/products/microelectrode-arrays/60pmea20030ir-ti</td></tr><tr><td>MEA amplifier</td><td>Multi Channel Systems, GmbH</td><td>MEA1060-Inv</td><td>http://www.multichannelsystems.com/products/mea1060-inv</td></tr><tr><td>Bottom perfusion groundplate for pMEA</td><td>Multi Channel Systems, GmbH</td><td>MEA1060-Inv-(BC)-PGP</td><td>http://www.multichannelsystems.com/products/mea1060-inv-bc-pgp</td></tr><tr><td>3-axis Motorized Micromanipulator</td><td>Sutter Instruments, Novato, CA</td><td>MP-285</td><td>https://www.sutter.com/MICROMANIPULATION/mp285.html</td></tr><tr><td>Micromanipulator Control System</td><td>Sutter Instruments, Novato, CA</td><td>MPC-200</td><td>https://www.sutter.com/MICROMANIPULATION/mpc200.html</td></tr><tr><td>Gantry style micromanipulator stand with linear slide</td><td>Sutter Instruments, Novato, CA</td><td>MT-75/LS</td><td>https://www.sutter.com/STAGES/mt75.html</td></tr><tr><td>8-channel Programmable Multichannel Pressure Injector</td><td>OEM: MicroData Instrument, S. Plainfield, NJ&lt;br/&gt; Vendor: Harvard Apparatus UK</td><td>PM-8000 or PM-8</td><td>OEM: http://www.microdatamdi.com/pm8000.htm&lt;br/&gt; Vendor: https://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/product_11555_10001_39808_-1&lt;br /&gt;_HAUK_ProductDetail</td></tr><tr><td>Axopatch 200A Integrating Patch Clamp Amplifier</td><td>Molecular Devices, Sunnyvale, CA</td><td>Axopatch 200A</td><td>Axopatch 200A has been replaced with a newer model Axopatch 200B:&lt;br/&gt; https://www.moleculardevices.com/systems/axon-conventional-patch-clamp/axopatch-200b-amplifier</td></tr><tr><td>Patch clamp headstage</td><td>Molecular Devices, Sunnyvale, CA</td><td>CV 201A</td><td>http://mdc.custhelp.com/app/answers/detail/a_id/16554/~/axopatch-200a%3A-selection-cv-headstage</td></tr><tr><td>Vacuum waste kit</td><td>ALA Scientific Instruments, Farmingdale, NY</td><td>VMK</td><td>http://alascience.com/product/vacuum-waste-kit/</td></tr><tr><td>Pipette holder</td><td>Warner Instruments, Hamden, CT</td><td>QSW-A10P</td><td>https://www.warneronline.com/product_info.cfm?id=915</td></tr><tr><td>Pre-pulled 10 &amp;mu;m tip diameter glass micropipettes</td><td>World Precision Instruments, Sarasota, FL</td><td>TIP10TW1</td><td>https://www.wpiinc.com/products/laboratory-supplies/make-selection-pre-pulled-glass-pipettes-plain/</td></tr><tr><td>Zoom stereomicroscope</td><td>Nikon, Tokyo, Japan</td><td>SMZ-745T</td><td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ745</td></tr><tr><td>Microscope boom stand with dual linear ball bearing arm</td><td>Old School Industries, Inc., Dacono, CO</td><td>OS1010H-16BB</td><td>http://www.osi-incorp.com/productdisplay/dual-linear-ball-bearing-arm</td></tr><tr><td>Zoom Stereo Microscope with C-LEDS Hybrid LED Stand</td><td>Nikon, Tokyo, Japan</td><td>SMZ-445</td><td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ445</td></tr><tr><td>Inverted microscope system</td><td>Nikon, Tokyo, Japan</td><td>Eclipse Ti-E</td><td>https://www.nikoninstruments.com/Products/Inverted-Microscopes/Eclipse-Ti-E</td></tr><tr><td>Ames medium</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A1420</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/a1420</td></tr><tr><td>L-Glutamic Acid (Glutamate)</td><td>Sigma-Aldrich, St. Louis, MO</td><td>G5667</td><td>http://www.sigmaaldrich.com/catalog/product/mm/100291</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich, St. Louis, MO</td><td>S8761</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/s8761</td></tr><tr><td>60 mm Petri dish (10 mm tall)</td><td>Fischer Scientific, Waltham, MA</td><td>FB0875713A</td><td>60 mm clear petri dish; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875713a</td></tr><tr><td>Jewelers #5 Forceps</td><td>World Precision Instruments, Sarasota, FL</td><td>555227F</td><td>https://www.wpiinc.com/products/laboratory-supplies/555227f-jewelers-5-forceps-11cm-straight-titanium/</td></tr><tr><td>Standard Scalpel Blad #24</td><td>World Precision Instruments, Sarasota, FL</td><td>500247</td><td>https://www.wpiinc.com/products/laboratory-supplies/500247-standard-scalpel-blade-24/</td></tr><tr><td>Scalpel Handle #4</td><td>World Precision Instruments, Sarasota, FL</td><td>500237</td><td>https://www.wpiinc.com/products/laboratory-supplies/500237-scalpel-handle-4-14cm/</td></tr><tr><td>Vannas Tubingen Dissection Scissors</td><td>World Precision Instruments, Sarasota, FL</td><td>503378</td><td>https://www.wpiinc.com/products/laboratory-supplies/503378-vannas-tubingen-scissors-8cm-straight-german-steel/</td></tr><tr><td>Nylon mesh kit</td><td>Warner Instruments, Hamden, CT</td><td>NYL/MESH</td><td>https://www.warneronline.com/product_info.cfm?id=1173</td></tr><tr><td>Harp slice grid</td><td>ALA Scientific Instruments, Farmingdale, NY</td><td>HSG-5AD</td><td>http://alascience.com/product/standard-harp-slice-grids/</td></tr><tr><td>Ag/AgCl reference electrode pellet</td><td>Multi Channel Systems, GmbH</td><td>P1060</td><td>http://www.multichannelsystems.com/products/p1060</td></tr><tr><td>4 Channel Valve Controlled Gravity Perfusion System</td><td>ALA Scientific Instruments, Farmingdale, NY</td><td>VC3-4xG</td><td>http://alascience.com/product/4-channel-valve-controlled-gravity-perfusion-system/</td></tr><tr><td>Zyla 5.5 sCMOS microscope camera</td><td>Andor Technology, Belfast, UK</td><td>Zyla 5.5 sCMOS</td><td>http://www.andor.com/scientific-cameras/neo-and-zyla-scmos-cameras/zyla-55-scmos</td></tr><tr><td>Silver wire (50 &amp;mu;m diameter)</td><td>Fischer Scientific, Waltham, MA</td><td>AA44461G5</td><td>https://www.fishersci.com/shop/products/silver-wire-0-05mm-0-002-in-dia-annealed-99-99-metals-basis-3/aa44461g5</td></tr><tr><td>Tygon microbore tubing (1.6 mm diameter)</td><td>Cole Parmer, Vernon Hills , IL</td><td>EW-06419-01</td><td>https://www.coleparmer.com/i/tygon-microbore-tubing-0-020-x-0-060-od-100-ft-roll/0641901</td></tr><tr><td>Tilting Tool Holder with Steel Cannula</td><td>ALA Scientific Instruments, Farmingdale, NY</td><td>TILTPORT</td><td>One each of these were utilized for top perfusion and suction; http://alascience.com/product/tilting-tool-holder-with-steel-cannula/</td></tr><tr><td>Roscolux #26 Light Red Filter Sheet</td><td>Rosco Laboratories Inc., 52 Harbor View, Stamford, CT</td><td>R2611</td><td>Manufacturer: http://us.rosco.com/en/products/catalog/roscolux&lt;br/&gt; Vendor: https://www.bhphotovideo.com/c/product/43957-REG/Rosco_RS2611_26_Filter_Light.html</td></tr><tr><td>Smith &amp;amp; Wesson Galaxy Red Flashlight</td><td>Smith &amp;amp; Wesson, 2100 Roosevelt Avenue, Springfield, MA</td><td>4588</td><td>Manufacturer: https://www.smith-wesson.com/&lt;br/&gt; Vendor: http://www.mypilotstore.com/mypilotstore/sep/4588</td></tr><tr><td>MC_Rack Software</td><td>Multi Channel Systems, GmbH</td><td>MC_Rack</td><td>http://www.multichannelsystems.com/software/mc-rack</td></tr><tr><td>Labview Software</td><td>National Instruments, Austin, TX</td><td>LabVIEW</td><td>http://www.ni.com/labview/</td></tr><tr><td>NIS-Elements: Basic Research Software</td><td>Nikon, Tokyo, Japan</td><td>NIS-Elements BR</td><td>https://www.nikoninstruments.com/Products/Software/NIS-Elements-Basic-Research</td></tr></tbody></table>

methodology for biomimetic chemical neuromodulation of rat retinas with the neurotransmitter glutamate in vitro

感光变性疾病通过视网膜感光细胞的逐渐丧失而导致无法挽回的失明。视网膜假体是一种新兴的治疗感光退行性疾病, 寻求恢复视力的人为刺激幸存的视网膜神经元, 希望能获得可理解的视觉感知的病人。目前的视网膜假体已证明是成功的恢复有限的视力患者使用一系列电极电刺激视网膜, 但面临巨大的物理障碍, 恢复高视力, 自然视力的病人。使用天然神经递质的化学神经是一种仿生替代电刺激, 可以绕过与使用电神经的视网膜假肢相关的基本限制。具体而言, 化学神经有可能恢复更多的自然视力与可比或更好的视觉视力患者通过注射极少量的神经递质, 相同的自然药物的交流使用的视网膜化学突触, 比当前的电子假肢更精细的分辨率。然而, 作为一个相对未被探索的刺激范例, 没有建立的协议为实现视网膜的化学刺激在体外。这项工作的目的是提供一个详细的框架, 以完成化学刺激视网膜的研究者谁希望研究的潜在化学调节的视网膜或类似的神经组织体外。在这项工作中, 我们描述的实验设置和方法, 诱导视网膜神经节细胞 (王国) 穗反应类似于视觉光反应的野生型和感光 wholemount 大鼠视网膜的注射控制体积神经递质谷氨酸进入视网膜空间使用玻璃 micropipettes 和定制多端口微流控装置。这种方法和协议是通用的, 足以适应调节使用其他神经递质或甚至其他神经系统组织。

该协议可用于化学刺激正常, 野生类型的视网膜和感光变性视网膜, 尽管大量的细胞重塑造成的损失的光。在开始实验, 无论是感光器退化或野生类型的视网膜, 记录和刺激设备 (图 1和图 2) 需要准备好, pMEA (图 5) 应该被清除, 以最小化每个电极通道上的噪音 (图 6)。虽然感光变性视网?...

Watch this Scientific Journal Video about Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro at JoVE.com

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

本议定书描述了一种新的方法, 研究一种形式的化学神经的 wholemount 大鼠视网膜体外与神经递质谷氨酸。化学神经是一种有前途的替代传统的电神经视网膜神经元治疗不可逆转性失明引起的感光变性疾病。

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

methodology-for-biomimetic-chemical-neuromodulation-rat-retinas-with

Research

JoVE Journal

Bioengineering

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

7.7K 次观看.  University of Illinois at Chicago. 本议定书描述了一种新的方法, 研究一种形式的化学神经的 wholemount 大鼠视网膜体外与神经递质谷氨酸。化学神经是一种有前途的替代传统的电神经视网膜神经元治疗不可逆转性失明引起的感光变性疾病。

视频: Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

科研

医学

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

神经科学

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

生物学

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

优化离 体 视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

评估视网膜神经节细胞层中的钙活性

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

作者聚焦:一种使用钙成像进行神经电刺激的创新方法 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

电刺激平装视网膜中的钙成像

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

摘要

探索更多视频

此视频中的章节

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

在Wholemount大鼠视网膜免疫组化和钙成像方法

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

视网膜平片准备成像和免疫组化优化方案

定量自发 Ca²⁺ 通量及其在原发性小鼠中脑神经元中的下游效应

小鼠视网膜的经瞳双光子体内成像

优化离体视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

电刺激平装视网膜中的钙成像

电刺激平装视网膜中的钙成像

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

摘要

探索更多视频

此视频中的章节

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

在Wholemount大鼠视网膜免疫组化和钙成像方法

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

视网膜平片准备成像和免疫组化优化方案

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

小鼠视网膜的经瞳双光子体内成像

优化离 体 视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

电刺激平装视网膜中的钙成像

电刺激平装视网膜中的钙成像

定量自发 Ca²⁺ 通量及其在原发性小鼠中脑神经元中的下游效应

优化离体视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能