نشكر الراحل رودي ليمبورغ لافتعال محيط الذراع كاردان، كيمبرلي جاميسون، ماثيو ماك هنري، وراجو Metherate لإقراض لنا المعدات، والمط كيلبر وكينتارو أريكاوا، للتشجيع. وأيد هذا العمل من قبل مؤسسة العلوم الوطنية (NSF) العليا زمالة أبحاث لKJM ومنحة جبهة الخلاص الوطني IOS-1257627 لبنك التنمية الآسيوي

الكتاب تعلن أنه ليس لديهم مصالح مالية المتنافسة.

تسجيل داخل الخلايا يمكن أن تكون تقنية صعبة لإتقان بسبب العديد من الخطوات التقنية المعنية. للتجارب ناجحة يجب أخذها بعين الاعتبار عدة نقاط هامة. أولا، من المهم أن يكون لديك جدول صحيح vibrationally معزولة التي يتم تنفيذ التجربة. استخدام العديد من الباحثين الجداول الهواء التي ...

ويلاحظ الخصائص الكهربائية للخلايا مثل الخلايا العصبية عن طريق قياس تدفق الأيونات عبر أغشية الخلايا على أنها تغير في الجهد أو التيار. وقد وضعت مجموعة متنوعة من التقنيات الكهربية لقياس الأحداث الكهربيولوجي في الخلايا. الخلايا العصبية الموجودة في عيون الحيوانات يمكن الوصول إليها والدوائر التي غالبا ما تكون أقل تعقيدا مما كانت عليه في الدماغ، مما يجعل هذه الخلايا مرشحين جيدين للدراسة الكهربية. وتشمل التطبيقات المشتركة للالكهربية في العين تخطيط كهربية الشبكية (أرج) 1،2 ومسرى مكروي تسجيل داخل الخلايا. أرج ينطوي على وضع القطب أو على العين من حيوان، وتطبيق حافز الضوء، وقياس التغير في التيار الكهربائي في شكل مبلغ من ردود جميع الخلايا المجاورة 3-6. إذا كان أحد يهتم تحديدا في وصف الحساسيات الطيفية للخلايا مستقبلة للضوء الفردية، وغالبا ما أنواع الخلايا متعددة تستجيب في وقت واحد في مختلف جوانب القوة لحافز معين؛ وبالتالي فإنهقد يكون من الصعب تحديد الحساسيات من أنواع معينة من الخلايا من البيانات أرج خاصة إذا كان هناك عدة أنواع مختلفة من خلايا مستقبلة للضوء طيفيا-مماثلة في العين. حل واحد محتمل هو خلق المعدلة وراثيا ذبابة الفاكهة مع مبصرة (أوبسين) الجينات في المصالح التي أعرب عنها في الخلايا غالبية R1-6 في العين ثم أداء ERGs 7. وتشمل العيوب المحتملة لهذه الطريقة لا للانخفاض في التعبير عن البروتين مستقبلة للضوء 8، والإطار الزمني الطويل لتوليد وفحص الحيوانات المعدلة وراثيا. للعيون مع أنواع أقل من المستقبلات الضوئية متميزة طيفيا، يمكن أن تكيف العين مع المرشحات الملونة تساعد في خفض مساهمة بعض أنواع الخلايا إلى أرج، مما يسمح تقدير حساسية الطيفية ماكسيما 9. تسجيل داخل الخلايا هو أسلوب آخر حيث يخوزق القطب غرامة خلية ويتم تطبيق التحفيز. سجلات القطب فقط أن لحاقاستجابة الخلية idual بحيث تسجيل من وتحليل الخلايا الفردية متعددة يمكن أن تسفر عن حساسيات معينة من أنواع مختلفة من الخلايا من الناحية الفسيولوجية 10-14. على الرغم من أن بروتوكول لدينا يركز على تحليل الحساسية الطيفية، والمبادئ الأساسية من بين الخلايا تسجيل مع أقطاب الحادة للتعديل لتطبيقات أخرى. باستخدام إعداد مختلفة من العينة، على سبيل المثال، وباستخدام أقطاب الكوارتز حادة، يمكن للمرء أن يسجل من أعمق في الفص البصري أو مناطق أخرى في الدماغ، وهذا يتوقف على السؤال المطروح. على سبيل المثال، وأوقات استجابة خلايا مستقبلة للضوء الفردية 15، نشاط الخلايا في البصرية فصوص 16 (الصفيحة، النخاع أو lobula 17)، الدماغ 18 أو غيرها من العقد 19 ويمكن أيضا أن تسجل مع تقنيات مشابهة، أو يمكن الاستعاضة مؤثرات اللون مع الاستقطاب 20 -22 أو الاقتراح محفزات 23،24. Phototransduction، العملية التي من خلالها الضوءيمتص الطاقة وتحويلها إلى إشارة الكهروكيميائية، هو سمة قديمة المشتركة إلى يوم تقريبا جميع الحاضرين شعب الحيوانات 25. الصباغ البصرية الموجودة في خلايا مستقبلة للضوء، ومسؤولا عن بدء phototransduction البصرية رودوبسين. مصنوعة رودوبسين في جميع الحيوانات تتكون من بروتين أوبسين، وهو عضو في 7 الغشاء G عائلة مستقبلات البروتين يقترن، وحامل اللون يرتبط وهي مشتقة من شبكية العين أو جزيء مشابه 26،27. أوبسين تسلسل الأحماض الأمينية وهيكل حامل اللون يؤثر على امتصاص رودوبسين لأطوال موجية مختلفة من الضوء. عندما يتم امتصاص الفوتون من قبل حامل اللون رودوبسين يصبح تفعيلها، وبدء سلسلة G-البروتين في الخلية الذي يؤدي في نهاية المطاف إلى فتح قنوات أيون بغشاء 28. وخلافا لمعظم الخلايا العصبية، وخلايا مستقبلة للضوء تخضع التغييرات المحتملة متدرجة التي يمكن قياسها على أنها تغير نسبي في السعة استجابة مع تغيير التحفيز خفيفة. وعادة ما تعطىنوع مبصرة يعبر فقط الجين أوبسين واحد (على الرغم من وجود استثناءات 8،10،29-31). متطورة رؤية الألوان، من النوع الذي يوجد في كثير من الفقاريات والمفصليات، ويتحقق مع العين معقدة من مئات أو آلاف من خلايا مستقبلة للضوء كل تعبير عن واحد أو أكثر من أنواع أحيانا رودوبسين. يتم التقاط المعلومات البصرية من خلال مقارنة الردود على فسيفساء مبصرة عبر معقدة الإشارات العصبية المصب في العين والدماغ، مما يؤدي إلى تصور صورة كاملة مع اللون والحركة. بعد قياس ردود الخام من خلية مستقبلة للضوء لأطوال موجية مختلفة من الضوء عبر تسجيل داخل الخلايا، فمن الممكن لحساب حساسيتها الطيفية. ويستند هذا الحساب على مبدأ Univariance، التي تنص على أن استجابة الخلايا المستقبلة للضوء هي تعتمد على عدد من الفوتونات أنها تمتص، ولكن ليس على خصائص معينة من الفوتونات أنها تمتص 32. أي الفوتون الذي absorbed بواسطة رودوبسين من شأنها أن تحفز نفس النوع من الاستجابة. في الممارسة العملية، وهذا يعني أن السعة استجابة الخام الخلية سيزيد إما بسبب زيادة في شدة الضوء (الفوتونات أكثر لاستيعاب)، أو إلى التحول في الطول الموجي نحو حساسية ذروته (احتمال أكبر من رودوبسين استيعاب أن الطول الموجي). علينا الاستفادة من هذا المبدأ في ربط الاستجابات الخلوية في كثافة معروفة ونفس الطول الموجي للردود على أطوال موجية مختلفة وبنفس الشدة ولكن الحساسية النسبية غير معروفة. وغالبا ما يتم تحديد أنواع الخلايا التي الطول الموجي الذي قمم حساسيتها. هنا نعرض طريقة واحدة لتسجيل داخل الخلايا وتحليل الحساسية الطيفية للخلايا مستقبلة للضوء في العين فراشة، مع التركيز على جعل هذه الطريقة أكثر سهولة للمجتمع العلمي على نطاق أوسع. وعلى الرغم من تسجيل داخل الخلايا لا تزال شائعة في الأدب، ولا سيما فيما يتعلق رؤية الألوان في الحشرات، وقد وجدنا ثار أوصاف المواد والأساليب وعادة ما تكون قصيرة جدا للسماح لاستنساخ هذه التقنية. نقدم هذه الطريقة في شكل شريط فيديو بهدف السماح أسهل النسخ المتماثل. وصفنا هذه التقنية أيضا استخدام المعدات التي يمكن الحصول عليها بسهولة وبأسعار معقولة. نعالج المحاذير الشائعة التي غالبا ما لا يتم الإبلاغ، التي تبطئ البحوث عند تحسين تقنية جديدة ومعقدة.

<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

تسجيل داخل الخلايا هي تقنية قوية تستخدم لتحديد كيف يمكن لخلية واحدة قد يستجيب لحافز معين. في بحث الرؤية، وقد تم تسجيل داخل الخلايا تاريخيا تقنية شائعة تستخدم لدراسة الحساسيات من خلايا مستقبلة للضوء الفردية للمؤثرات الضوء المختلفة التي لا تزال تستخدم اليوم. ومع ذلك، لا تزال هناك ندرة في منهجية مفصلة في الأدب للباحثين الذين يرغبون في تكرار التجارب تسجيل داخل الخلايا في العين. هنا نقدم الحشرة كنموذج لدراسة علم وظائف الأعضاء العين بشكل عام. وتقع خلايا مستقبلة للضوء الحشرات بالقرب من سطح العين، وبالتالي من السهل الوصول إليها، والعديد من الآليات التي تشارك في الرؤية وحفظها عبر شعب الحيوانات. نحن تصف الإجراء الأساسي في الجسم الحي تسجيل داخل الخلايا من خلايا مستقبلة للضوء في العين من فراشة، وذلك بهدف جعل هذه التقنية في متناول الباحثين لديهم خبرة سابقة قليلا في البريدlectrophysiology. ونحن نقدم المعدات الأساسية اللازمة، وكيفية إعداد فراشة حية للتسجيل، وكيفية إدراج مسرى مكروي الزجاج في خلية واحدة، وأخيرا إجراءات تسجيل نفسها. نحن أيضا شرح التحليل الأساسي للبيانات استجابة أولية لتحديد حساسية الطيفية من أنواع الخلايا الفردية. على الرغم من أن بروتوكول لدينا يركز على تحديد حساسية الطيفية، منبهات أخرى (على سبيل المثال، الضوء المستقطب) والاختلافات في طريقة قابلة للتطبيق لهذا الإعداد.

بالنسبة لكثير من عناصر الإعداد التسجيل، وصفا مكتوبا لا توفر ما يكفي من التفاصيل. الشكل رقم 1 هو التخطيطي من المكونات الداخلة في الإعداد التسجيل الكامل. في الشكل 2، يتم رسم أطياف الضوء الأبيض وكل مرشح التدخل لإعطاء معنى لماذا هناك حاجة إلى معامل ...

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

وأظهرت هذه التقنية الكهربية من تسجيل داخل الخلايا وتستخدم لتحديد الحساسيات الطيفية للخلايا مستقبلة للضوء واحدة في العين مجمع فراشة.

تحديد مبصرة خلية الحساسية الطيفية في نموذج الحشرات من<em&gt; في فيفو</em&gt; بين الخلايا تسجيلات

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

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Neuroscience

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

11.4K Views.  University of California, Irvine.  وأظهرت هذه التقنية الكهربية من تسجيل داخل الخلايا وتستخدم لتحديد الحساسيات الطيفية للخلايا مستقبلة للضوء واحدة في العين مجمع فراشة. 

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.

تشريح والثقافة من الخلايا العصبية من النخاع الشوكي صواري الجنينية

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

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.

وحدة واحدة في الجسم الحي تسجيلات من chiasm البصرية من الجرذ

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.

توصيف بالسكان خلية كهربية من GFP - يعرب عن السليمة في الشبكية

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.

الفلورسنت وصفها الوراء يسمح لخارج الخلية المستهدفة واحدة وحدة التسجيل من الخلايا العصبية التي تم تحديدها<em&gt; في الجسم الحي</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.

الطريقة الكهربية لالردود تسجيل بين الخلايا الجهد ل<em&gt; ذبابة الفاكهة</em&gt; خلايا مستقبلة للضوء وInterneurons إلى النور المحفزات<em&gt; في فيفو</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.

طريقة الكهربية لالكامل خلية الجهد المشبك تسجيلات من<em&gt; ذبابة الفاكهة</em&gt; المستقبلات الضوئية

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.

أزاله الحساسية والتعافي من المستقبلات الضوئية جراد الصيد عند تسليم التحفيز الخفيفة

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;

الطرق الكهروفسيولوجية لقياس مستويات التصبغ الضوئي في مستقبلات ذبابة الفاكهة الضوئية

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.

في الجسم الحي التسجيل داخل الخلايا للحساسية الطيفية للخلايا المستقبلة للضوء في فراشة

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 ...

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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.

التسجيل الفيزيولوجي الكهربي لاستجابات الجهد لذبابة الفاكهة المستقبلات الضوئية لشبكية العين لمحفزات الضوء

1. Recording from R1-R6 Photoreceptors 

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

تحديد مبصرة خلية الحساسية الطيفية في نموذج الحشرات من

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