Biz teşvik için, bize ekipman kredi için kardan kol çevresini, Kimberly Jamison, Matthew McHenry ve Raju Metherate imalatı için geç Rudy Limburg teşekkür ve Almut Kelber ve Kentaro Arikawa. Bu çalışma ADB için bir Ulusal Bilim Vakfı (NSF) Bilimsel Araştırma KJM için Bursu ve NSF hibe IOS-1257627 tarafından desteklenen

Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Hücre içi kayıt nedeniyle ilgili pek çok teknik adımlara ana zor bir teknik olabilir. Başarılı deneyler için birkaç önemli noktalar dikkate alınmalıdır. İlk olarak, deney gerçekleştirilmiştir edildiği bir düzgün titreşimli izole tablo önemlidir. Birçok araştırmacı, tamamen üstün titreşim yalıtımı sağlayan, tabandan masanın üzerinde ayrı hava tabloları kullanın. Bizim kurulum mikromanipülatör / elektrot tutucu / numune sahne cihazı yerleştirildiği üstüne bir sandbox, kalın b...

nöronlar gibi hücrelerinin elektriksel özellikleri voltaj ve akım bir değişiklik olarak hücre zarlarından iyon akışını ölçmek sureti ile görülür. elektrofizyolojik teknikler çeşitli hücrelerde biyo etkinliklerini ölçmek için geliştirilmiştir. hayvanların gözlerine bulunan Nöronlar erişilebilir ve devre elektrofizyolojik çalışma için bu hücreleri iyi adaylar yapım, beyinde genellikle daha az karmaşıktır. Gözde elektrofizyoloji yaygın uygulamalar elektroretinografi (ERG) 1,2 ve mikroelektrot hücre içi kayıt bulunmaktadır. ERG, ya da bir hayvanın gözüne bir elektrot yerleştirme hafif bir uyan uygulamak ve yakındaki hücrelerin 3-6 tepkilerin bir toplamı olarak gerilim değişikliği ölçülmesini kapsar. tek bir kişi fotoreseptör hücrelerinin spektral hassasiyetlerini karakterize, özellikle ilgilenen varsa, genellikle birden fazla hücre tipleri aynı anda belirli bir uyarana farklı güçlerde de tepki; böylece onugözde spektral-benzer fotoreseptör hücrelerinin birkaç farklı türü vardır özellikle ERG verileri belirli hücre tiplerinin hassasiyetlerini belirlemek zor olabilir. Bir potansiyel çözelti göz çoğunluğu R1-6 hücrelerinde eksprese ilgi fotoreseptör (opsin) geni ile Drosophila oluşturmak ve sonra ERGler 7 yapmaktır. Bu yöntemin olası sakıncaları fotoreseptör protein 8 düşük ifadesine hayır ve transgenik hayvan üretimi ve tarama için uzun bir zaman dilimi bulunmaktadır. Spektral farklı fotoreseptör daha az çeşit gözlerde için, renkli filtreler ile gözün uyum böylece ERG bazı hücre tiplerinin katkı düşürücü spektral duyarlılık maxima 9 tahminini izin yardımcı olabilir. Hücre içi kayıt ince bir elektrot bir hücreyi impales başka tekniktir ve bir uyarıcı uygulanır. elektrot kayıtları sadece o individual hücrenin tepkisi kayıt ve çoklu bireysel hücrelerin analiz fizyolojik farklı hücre tipleri 10-14 spesifik hassasiyetlerini verim böylece. Bizim protokol spektral duyarlılık analizi üzerinde duruluyor olsa da, keskin elektrotlar ile kayıt hücre içi temel ilkeleri diğer uygulamalar için değiştirilebilir vardır. Örneğin, bir numune farklı hazırlanmasını kullanarak, ve keskin kuvars elektrotlar kullanılarak, tek bir isteniyor soruya bağlı olarak, beyindeki optik lob veya diğer bölgelerde daha derin kayıt olabilir. Örneğin, bireysel fotoreseptör hücrelerinin 15 yanıt süreleri, optik hücre aktivitesi 19 da benzer teknikler ile kaydedilebilir veya renk uyaranlar kutuplaşma 20 ile değiştirilir olabilir 16 (lamina, medullayı veya lobula 17), beyin 18 veya diğer ganglion loblar -22 veya hareket 23,24 uyaranlara. Fototransdüksiyon, süreç hangi ışık tarafındanEnerji absorbe edildi ve bir elektrokimyasal sinyaline dönüştürülür, hemen hemen tüm bugünkü hayvan fila 25 ortak eski bir özelliktir. fotoreseptör hücrelerinde bulunan ve görsel fototransdüksiyon başlatmaktan sorumludur görsel pigmenti rodopsin olduğunu. Tüm hayvanlarda Rhodopsins retina veya benzer molekül 26,27 türetilen bir opsin proteini, 7 transmembran G-protein kenetli reseptör ailesinin bir üyesi ve bir birleşik kromoforun oluşur. Amino asit dizisi ve kromofor yapısı opsin ışığın farklı dalgaboylarına karşı rodopsin absorbansı etkiler. Foton kromofor tarafından absorbe edildiğinde rodopsin sonuçta zara bağlı iyon kanalları 28 açılmasına neden olur hücrede bir G-proteini başlatarak, aktif hale gelir. En nöronlar farklı olarak, fotoreseptör hücrelerinin ışık uyarısına cevap değişen amplitüdünde görece bir değişiklik olarak ölçülebilir kademeli potansiyel değişimler geçirirler. Tipik olarak, belirli birfotoreseptör tipi tek opsin genini ifade (istisnalar 8,10,29-31 mevcut olsa da). Sofistike renk görme, birçok omurgalılarda ve eklembacaklılar bulunan türden, her biri veya bazen daha fazla rodopsin türlerini ifade fotoreseptör hücrelerinin yüzlerce ya da binlerce karmaşık bir gözle elde edilir. Görsel bilgi renk ve hareket ile tam bir görüntünün algılanması sonucu, göz ve beyinde karmaşık aşağı nöral sinyalizasyonu ile fotoreseptör mozaik üzerinde yanıtları karşılaştırarak yakalanır. hücre içi kayıt yoluyla ışığın farklı dalga boylarında bir fotoreseptör hücre ham tepkilerini ölçmek sonra, onun spektral duyarlılığı hesaplamak mümkündür. Bu hesaplama fotoreseptör hücre yanıtı ancak 32 emer fotonların belirli özelliklerine, o emer fotonların sayısına bağlı olduğunu ifade Univariance İlkesi dayanmaktadır. Abso olan herhangi bir fotontepkiyi neden olacaktır rodopsin tarafından rbed. Uygulamada, bu hücrenin ham tepki genlik (rodopsin yüksek olasılık olduğu dalga boyu emici) (absorbe daha fazla foton) ışık şiddeti bir artış ya bağlı artış, ya da zirveye hassasiyeti doğru dalga boyunda bir kaymaya anlamına gelir. Biz bilinen yoğunluğu ve farklı dalga boylarında tepkiler aynı dalga boyunda ve aynı yoğunlukta ama bilinmeyen bağıl duyarlılık hücresel yanıtları ile ilgili bu prensip faydalanmak. Hücre tipleri genellikle dalga boyunda kendi duyarlılık doruklarına tarafından tespit edilir. Burada geniş bir araştırma topluluğuna bu yöntem daha erişilebilir hale odaklanarak, hücre içi kayıt ve kelebek gözünde fotoreseptör spektral duyarlılık analizi için bir yöntem göstermektedir. Hücre içi kayıt, özellikle böcekler renk görme ile ilgili olarak, literatürde yaygın kalmakla birlikte, Tha buldukmateryal ve yöntemler t açıklamaları genellikle tekniğin yeniden üretimi için izin vermek için çok kısa bulunmaktadır. Biz onun daha kolay çoğaltma izin amacı ile video formatında bu yöntem mevcut. Biz de kolay elde ve uygun fiyatlı ekipmanlar kullanılarak tekniği açıklar. Biz yeni ve karmaşık teknik optimize ederken araştırma yavaşlatmak sık sık rapor değil ortak uyarılar, adres.

<table><tbody><tr><td>Butterfly pupae</td><td></td><td></td><td>Several local species available, need USDA permits for shipping. Carolina Bio Supply has several insect species that may be ordered within the U.S. without the need for additional permits</td></tr><tr><td>Large plastic cylinder</td><td></td><td></td><td>Any chamber that remains humidified will work</td></tr><tr><td>Insect pins, size 2</td><td>BioQuip</td><td>1208B2</td><td></td></tr><tr><td>100% Desert Mesquite Honey</td><td>Trader Joe&#039;s</td><td></td><td>Any honey or sucrose solution will work</td></tr><tr><td>Xenon Arc Lamp</td><td>Oriel Instruments</td><td>66003</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Universal Power Supply</td><td>Oriel Instruments</td><td>68805</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Optical Track</td><td>Oriel Instruments</td><td>11190</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Large (2x)</td><td>Oriel Instruments</td><td>11641</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Small (4x)</td><td>Oriel Instruments</td><td>11647</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Thread Adaptor, 8-32 Male to 1/4-20 Male, pack of 10</td><td>Newport Corporation</td><td>TA-8Q20-10</td><td></td></tr><tr><td>Optical Mounting Post, 1.0 in., 0.5 in. Dia. Stainless, 8-32 &amp;amp; 1/4-20 (5x)</td><td>Newport Corporation</td><td>SP-1</td><td></td></tr><tr><td>No Slip Optical Post Holder, 2 in., 0.5 in. Diameter Posts, 1/4-20 (5x)</td><td>Newport Corporation</td><td>VPH-2</td><td></td></tr><tr><td>Fixed lens mount, 50.8 mm</td><td>Newport Corporation</td><td>LH-2</td><td></td></tr><tr><td>Fixed lens mount, 25.4 mm</td><td>Newport Corporation</td><td>LH-1</td><td></td></tr><tr><td>Condenser lens assembly</td><td>Newport Corporation</td><td>60006</td><td></td></tr><tr><td>Convex silica lens, 50.8 mm</td><td>Newport Corporation</td><td>SPX055</td><td></td></tr><tr><td>Six Position Filter Wheel, x2</td><td>Newport Corporation</td><td>FW1X6</td><td></td></tr><tr><td>Filter Wheel Mount Hub</td><td>Newport Corporation</td><td>FWM</td><td></td></tr><tr><td>Concave silica lens, 25.4 mm</td><td>Newport Corporation</td><td>SPC034</td><td></td></tr><tr><td>Collimator holder</td><td>Newport Corporation</td><td>77612</td><td></td></tr><tr><td>Collimating beam probe</td><td>Newport Corporation</td><td>77644</td><td></td></tr><tr><td>Ferrule Converter, SMA Termination to 11 mm Standard Ferrule</td><td>Newport Corporation</td><td>77670</td><td>This adapter allows the fiber optic to fit into the collimator holder&amp;nbsp;</td></tr><tr><td>600 &amp;mu;m diameter UV-vis fiber obtic cable</td><td>Oriel Instruments</td><td>78367</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Shutter with drive unit</td><td>Uniblitz</td><td>100-2B</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.1 OD</td><td>Newport</td><td>FRQ-ND01</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.3 OD</td><td>Newport</td><td>FRQ-ND03</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.5 OD</td><td>Newport</td><td>FRQ-ND05</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 1.0 OD</td><td>Newport</td><td>FRQ-ND10</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 2.0 OD</td><td>Newport</td><td>FRQ-ND30</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 3.0 OD</td><td>Newport</td><td>FRQ-ND50</td><td></td></tr><tr><td>LS-1-Cal lamp</td><td>Ocean Optics</td><td>LS-1-Cal</td><td></td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>USB-2000</td><td></td></tr><tr><td>SpectraSuite Software</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>Interference bandpass filter, 300 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67749</td><td></td></tr><tr><td>Interference bandpass filter, 310 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67752</td><td></td></tr><tr><td>Interference bandpass filter, 320 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67754</td><td></td></tr><tr><td>Interference bandpass filter, 330 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67756</td><td></td></tr><tr><td>Interference bandpass filter, 340 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65614</td><td></td></tr><tr><td>Interference bandpass filter, 350 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67757</td><td></td></tr><tr><td>Interference bandpass filter, 360 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67760</td><td></td></tr><tr><td>Interference bandpass filter, 370 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67761</td><td></td></tr><tr><td>Interference bandpass filter, 380 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67762</td><td></td></tr><tr><td>Interference bandpass filter, 390 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67763</td><td></td></tr><tr><td>Interference bandpass filter, 400 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65732</td><td></td></tr><tr><td>Interference bandpass filter, 410 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65619</td><td></td></tr><tr><td>Interference bandpass filter, 420 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65621</td><td></td></tr><tr><td>Interference bandpass filter, 430 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65622</td><td></td></tr><tr><td>Interference bandpass filter, 440 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67764</td><td></td></tr><tr><td>Interference bandpass filter, 450 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65625</td><td></td></tr><tr><td>Interference bandpass filter, 460 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67765</td><td></td></tr><tr><td>Interference bandpass filter, 470 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65629</td><td></td></tr><tr><td>Interference bandpass filter, 480 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65630</td><td></td></tr><tr><td>Interference bandpass filter, 492 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65633</td><td></td></tr><tr><td>Interference bandpass filter, 500 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65634</td><td></td></tr><tr><td>Interference bandpass filter, 510 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65637</td><td></td></tr><tr><td>Interference bandpass filter, 520 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65639</td><td></td></tr><tr><td>Interference bandpass filter, 532 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65640</td><td></td></tr><tr><td>Interference bandpass filter, 540 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65642</td><td></td></tr><tr><td>Interference bandpass filter, 550 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65644</td><td></td></tr><tr><td>Interference bandpass filter, 560 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67766</td><td></td></tr><tr><td>Interference bandpass filter, 570 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67767</td><td></td></tr><tr><td>Interference bandpass filter, 580 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65646</td><td></td></tr><tr><td>Interference bandpass filter, 589 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65647</td><td></td></tr><tr><td>Interference bandpass filter, 600 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65648</td><td></td></tr><tr><td>Interference bandpass filter, 610 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65649</td><td></td></tr><tr><td>Interference bandpass filter, 620 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65650</td><td></td></tr><tr><td>Interference bandpass filter, 632 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65651</td><td></td></tr><tr><td>Interference bandpass filter, 640 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65653</td><td></td></tr><tr><td>Interference bandpass filter, 650 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65655</td><td></td></tr><tr><td>Interference bandpass filter, 660 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67769</td><td></td></tr><tr><td>Interference bandpass filter, 671 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65657</td><td></td></tr><tr><td>Interference bandpass filter, 680 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67770</td><td></td></tr><tr><td>Interference bandpass filter, 690 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65659</td><td></td></tr><tr><td>Interference bandpass filter, 700 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67771</td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Any metal structure will work that can be grounded and that fits the experimental setup.</td></tr><tr><td>Stereomicroscope, 6X, 12X, 25X, 50X magnification</td><td>Wild Heerbrugg</td><td>Wild M5</td><td>Any Stereomicroscope will do</td></tr><tr><td>Microscope stand with swinging arm and heavy base</td><td>McBain Instruments</td><td></td><td>Any heavy base with arm will do</td></tr><tr><td>Cardan arm</td><td></td><td></td><td>Custom built, See &lt;strong&gt;Figure 4&lt;/strong&gt;</td></tr><tr><td>Fiber-lite high intensity illuminator</td><td>Dolan-Jenner</td><td>MI-150</td><td>For lighting specimen</td></tr><tr><td>Fiber-lite goose-neck light guide</td><td>Dolan-Jenner</td><td>EEG 2823</td><td>Any goose-neck light guide will do</td></tr><tr><td>Marble table</td><td></td><td></td><td></td></tr><tr><td>Raised wooden table</td><td></td><td></td><td>Hole should be cut through this table so that the sandbox can rest on the marble table underneath</td></tr><tr><td>Wooden box filled with sand</td><td></td><td></td><td>custom built, any box with sand</td></tr><tr><td>Manipulator</td><td>Carl Zeiss - Jena</td><td></td><td></td></tr><tr><td>Electrode holder</td><td></td><td></td><td></td></tr><tr><td>Specimen stage</td><td></td><td></td><td></td></tr><tr><td>Alligator clip wires for grounding</td><td></td><td></td><td></td></tr><tr><td>Insulated copper wire</td><td></td><td></td><td></td></tr><tr><td>Silver wire, 0.125 mm diameter</td><td>World Precision Instruments</td><td>AGW0510</td><td></td></tr><tr><td>BNC cables</td><td></td><td></td><td></td></tr><tr><td>Preamplifier with headstage</td><td>Dagan Corporation</td><td>IX2-700</td><td></td></tr><tr><td>Humbug Noise reducer</td><td>Quest Scientific</td><td>Humbug</td><td></td></tr><tr><td>Oscilloscope, 30 MHz, 2 CH, Dual Trace, Alt-triggering, without probe</td><td>EZ Digital</td><td>os-5030</td><td></td></tr><tr><td>BNC T-adapter</td><td></td><td></td><td></td></tr><tr><td>Powerlab hardware 2/20</td><td>ADI instruments</td><td>ML820</td><td></td></tr><tr><td>Labchart software</td><td>ADI instruments</td><td>Chart 5</td><td></td></tr><tr><td>10 MHz Pulse Generator</td><td>BK Precision</td><td>4030</td><td></td></tr><tr><td>Glass pipette puller</td><td>Sutter Instruments</td><td>P-87</td><td></td></tr><tr><td>Borosillicate glass capillaries with filament</td><td>World Precision Instruments</td><td>1B120F-4</td><td></td></tr><tr><td>Potassium chloride, 3 M</td><td></td><td></td><td></td></tr><tr><td>Slotted plastic tube</td><td></td><td></td><td></td></tr><tr><td>Low melting temperature wax</td><td></td><td></td><td></td></tr><tr><td>Soldering Iron</td><td>Weller</td><td></td><td></td></tr><tr><td>Platform with ball-and-socket magnetic base</td><td>Hama photo and video</td><td></td><td></td></tr><tr><td>Double edge carbon steel, breakable razor blade</td><td>Electron Microscopy Sciences</td><td>72004</td><td></td></tr><tr><td>Vaseline</td><td></td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

determination of photoreceptor cell spectral sensitivity in an insect model from in vivo intracellular recordings

Hücre içi kayıt tek bir hücre belirli bir uyarana tepki nasıl belirlemek için kullanılan güçlü bir tekniktir. vizyon araştırmada, hücre içi kayıt tarihsel bugün hala kullanılmakta olan farklı ışık uyaranlara karşı bireysel fotoreseptör hücrelerinin hassasiyetlerini incelemek için kullanılan ortak bir teknik olmuştur. Ancak, göz hücre içi kayıt deneyleri çoğaltmak isteyen araştırmacılar için literatürde detaylı metodoloji bir eksiklik kalır. Burada daha genel göz fizyolojisini incelemek için bir model olarak böceği sunuyoruz. Böcek fotoreseptör hücreleri gözün yüzeyine yakın bulunan ve bu nedenle ulaşmak kolay ve vizyonu katılan mekanizmaların birçok hayvan filumlarında boyunca korunduğu vardır. Biz e az önce deneyimi olan araştırmacılara bu teknik daha erişilebilir hale getirmek amacı ile, bir kelebeğin gözünde fotoreseptör hücrelerinin in vivo hücre içi kayıt için temel prosedür açıklanmaktadırlectrophysiology. Biz tek bir hücreye bir cam mikroelektrot ve nihayet kayıt işlemini kendisi nasıl takılacağını, kayıt için canlı kelebek nasıl hazırlanacağını, gerekli temel donanımları tanıtmak. Biz de bireysel hücre tiplerinin spektral duyarlılığı belirlemek için ham tepki verilerinin temel analizini açıklar. Bizim protokol spektral duyarlılığı belirleyen odaklanır rağmen, diğer uyaranlara (örneğin, polarize ışık) ve yöntemin varyasyonları bu kurulum için geçerlidir.

Kayıt kurulum birçok unsuru, bir yazılı açıklama yeterli ayrıntı sağlamaz. Şekil 1 tam kayıt kurulumu katılan bileşenlerin şematik olarak göstermektedir. Şekil 2&#39;de, spektrumları bir düzeltme faktörü gereklidir neden sens ve ne bu düzeltme hesaplamak için gerekli olan vermek üzere, beyaz bir ışık ve her bir girişim filtresi için çizilir. 3 fotoğraf ve bunlar için kullanılan kardan kolun bir diyagramını göstermekted...

Watch this Scientific Journal Video about Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings at JoVE.com

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

hücre içi kayıt elektrofizyolojik teknik gösterdi ve bir kelebeğin bileşik gözünde tek fotoreseptör hücrelerinin spektral hassasiyetlerini belirlemek için kullanılır.

bir Böcek Modeli fotoreseptör hücre spektral Duyarlılık Belirlenmesi<em&gt; İn Vivo</em&gt; Hücre içi Kayıtlar

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

determination-photoreceptor-cell-spectral-sensitivity-an-insect-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

11.3K Görüntüleme.  University of California, Irvine.  hücre içi kayıt elektrofizyolojik teknik gösterdi ve bir kelebeğin bileşik gözünde tek fotoreseptör hücrelerinin spektral hassasiyetlerini belirlemek için kullanılır. 

Video: Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Tek birim In vivo</emSıçan, optik kiazma> Kayıtlar

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Bozulmamış Retina Elektrofizyolojik Karakterizasyonu GFP-ifade eden hücre popülasyonlarının

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Recording Hücre içi Gerilim Yanıtları için Elektrofizyolojik Yöntem<em&gt; Drosophila</em&gt; Işık Uyaranların için Fotoreseptörler ve internöronlar<em&gt; İn Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Tam Hücreli Gerilim Kelepçesi Kayıtları için Elektrofizyolojik Yöntem<em&gt; Drosophila</emPhotoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

Bir Işık Uyaranteslimi Üzerine Kerevit Fotoreseptörlerinin Duyarsızlaştırılması ve İyileştirilmesi

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Drosophila Fotoreseptörlerinde Fotopigment Düzeylerini Ölçmek için Elektrofizyolojik Yöntemler

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

In Vivo Intracellular Recording of Photoreceptor Cell Spectral Sensitivity in a Butterfly

Source: McCulloch, K. J., et al.. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53829">Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly and determining the spectral sensitivity of individual cell types. This technique enables the study of sensitivities of individual photoreceptor cells to different light stimuli.

Bir Kelebekte Fotoreseptör Hücre Spektral Duyarlılığının İn Vivo Hücre İçi Kaydı

1. Specimen Prep and Recording Procedure

	Prepare the Specimen
	
		Affix an individual butterfly inside a small plastic tube with hot wax so the head is ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

Source: Juusola, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54142">Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.</a> J. Vis. Exp. (2016).
This video outlines the protocol for electrophysiological recording of photoreceptor activity in the eye of an immobilized Drosophila, enabling precise measurement of voltage changes in response to light stimuli.

Drosophila Retinal Fotoreseptörlerinin Işık Uyaranlarına Voltaj Yanıtlarının Elektrofizyolojik Kaydı

1. Recording from R1-R6 Photoreceptors 

	Always be grounded when operating the microelectrode amplifier (for example by touching the metal surface of the ...

bir Böcek Modeli fotoreseptör hücre spektral Duyarlılık Belirlenmesi

Özet

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Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Tek birim In vivo Kayıtlar

Bozulmamış Retina Elektrofizyolojik Karakterizasyonu GFP-ifade eden hücre popülasyonlarının

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir

Recording Hücre içi Gerilim Yanıtları için Elektrofizyolojik Yöntem

Tam Hücreli Gerilim Kelepçesi Kayıtları için Elektrofizyolojik Yöntem

Bir Işık Uyaranteslimi Üzerine Kerevit Fotoreseptörlerinin Duyarsızlaştırılması ve İyileştirilmesi

Drosophila Fotoreseptörlerinde Fotopigment Düzeylerini Ölçmek için Elektrofizyolojik Yöntemler

Bir Kelebekte Fotoreseptör Hücre Spektral Duyarlılığının İn Vivo Hücre İçi Kaydı

Drosophila Retinal Fotoreseptörlerinin Işık Uyaranlarına Voltaj Yanıtlarının Elektrofizyolojik Kaydı