وأيد هذا العمل من قبل NRSA 1F31DC012985 منحة دكتوراه مسبقا (لRD) ومنح المعاهد الوطنية للصحة لJC نود أن نشكر الدكتور ينيا فايس لتعليقات مفيدة على المخطوطة، والدكتور ريان جوزيف لشخصيات مساعدة تجميع، والدكتور فريدريك ماريون-الإستطلاع للحصول على المشورة التقنية مفيدة. نود أيضا أن نعترف تعليقات مفيدة من أربعة المعلقين.

<ol>
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والكتاب ليس لديهم ما يكشف.

الشعيرات Labellar تختلف في سهولة تسجيل بسبب الاختلافات في التشكل وتنظيم التشريحية. أحيانا لا يستجيب إلى أي sensillum وtastants، حتى واحد ما هو معروف للحصول على رد إيجابي. التردد الذي يحدث هذا يختلف تبعا لنوع sensillum و. L الشعيرات هي الأكثر استجابة باستمرار وسهلة نسبيا للوصول بسبب طو...

حاسة التذوق يسمح حشرة للكشف عن مجموعة واسعة من المواد الكيميائية القابلة للذوبان، ويلعب دورا هاما في قبول مادة مغذية، أو رفض واحد الضارة أو السامة. ويعتقد الذوق أيضا للعب دور في اختيار الشريك، من خلال الكشف عن الفيرومونات 1-5. جعلت هذه الوظائف الهامة والمتنوعة النظام طعم الحشرات هدفا مقنعة للتحقيق في كيفية النظم الحسية ترجمة الاشارات البيئية في المخرجات السلوكية ذات الصلة. الوحدة الأساسية للنظام طعم البطن ذبابة الفاكهة هو الشعر والذوق، أو sensillum و. جزيئات دخول sensillum وعبر المسام في 2،6 طرفها. تم العثور على الشعيرات labellum، والساقين، وهامش الجناح، والبلعوم 6. على labellum، والنمطية عدد وموقع الشعيرات. هناك ثلاث فئات من الشعيرات المورفولوجية بناء على طول: طويل (L) والمتوسطة (I)، وقصيرة (S<html> ) الشعيرات 7،8. يحتوي كل sensillum وإما اثنين (I-نوع) أو أربعة (L-S-نوع و) الخلايا العصبية مستقبلات الذوقية (GRNs) 9. GRNs مختلفة الرد على فئات مختلفة من المحفزات طعم: المر والسكر والملح والأسمولية 7،10 والتعبير عن مجموعات فرعية مختلفة من المستقبلات الذوقية 8،11-13. فقط أنا وS-نوع الشعيرات تحتوي GRNs المر استجابة 8،10. يتم ترحيل المشروع GRNs إلى العقدة تحت المريء (مجموعة العمليات الخاصة) والتنشيط من خلال جزيئات الذوق إلى الجهاز العصبي المركزي العالي لفك التشفير، مما أدى إلى استجابة سلوكية 6. عدد قليل نسبيا من الخلايا العصبية وقابليته للتحليل الجزيئي والسلوكية جعل النظام طعم ذبابة الفاكهة نموذجا ممتازا للتحقيق في أنظمة الذوقية بشكل عام. السهولة النسبية التي يمكن التلاعب بها النظام عبر طفرة جينية أو نظام التعبير GAL4-UAS أيضا بمثابة أداة قيمة 14،15. ontent &quot;&gt; لأن هذه الشعيرات تبرز من سطح labellum، لأنها تجعل أهداف ممتازة للالكهربية. اطلاق النار من GRNs يمكن رصدها باستخدام تسجيل خارج الخلية. تاريخيا، وطريقة تسجيل جنبا إلى الجدار، والذي يستخدم الزجاج الكهربائي إدراجها في sensillum وتسجيل نشاط الخلايا العصبية، وقد استخدم 26، ولكن هذا الأسلوب يمثل تحديا تقنيا على القيام بها، وأنه من الصعب أن يسجل لفترة طويلة من كل إعداد. طريقة غيض تسجيل، والذي يقيس استجابة الخلايا العصبية مع القطب أن في وقت واحد يسلم منبهة للذوق، وأصبح منذ الأسلوب المفضل 9،16. وقد استخدمت وللتحقيق في نظام طعم ذبابة الفاكهة السوداء البطن 8،10،17،18 فضلا عن عدد من أنواع الحشرات الأخرى 19-23. له تم سهلت كثيرا من خلال تطوير مكبر للصوت tastePROBE، التي تغلبت احدة من العوائق الرئيسية لطريقة التسجيل عن طريق بلاغ للتعويضفرق الجهد الكبير بين الإلكترود المرجعي وsensillum والحشرات، مما يتيح للإمكانات العمل GRN ليتم تسجيلها دون التضخيم المفرط أو التصفية 24. وثمة تطور مهم آخر استخدام tricholine سترات كما بالكهرباء يسجل 25. TCC يقمع الاستجابات من GRN الحساسة للالأسمولية ولا تحفيز GRN الحساسة الملح، مما يجعل الاستجابات التي تم إنشاؤها بواسطة tastants المر والسكر أسهل بكثير لتحليل 25. نحن هنا تصف كيف يتم تنفيذ تسجيل غيض من ذبابة الفاكهة labellar الشعيرات حاليا في المختبر كارلسون. وهذا البروتوكول شرح كيفية تأسيس منصة مناسبة الكهربية، وكيفية إعداد الطاير، وكيفية تنفيذ تسجيلات الذوق. كما نقدم بعض البيانات التي تم الحصول عليها عن طريق تسجيل ممثل من مجموعات فرعية من ذبابة الفاكهة الشعيرات، وكذلك بعض القضايا المشتركة والحلول المحتملة التي يمكن مواجهتها عند استخدام هذاالتقنية.

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	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:168px;">Stereo Zoom Microscope</td>
			<td style="width:187px;">Olympus&#160;</td>
			<td style="width:211px;">SZX12 DFPLFL1.6x PF eyepieces: WHN10x-H/22</td>
			<td style="width:219px;">capable of ~150x magnification with long working distance table mount stand</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Anti-vibration Table</td>
			<td style="width:187px;">Kinetic Systems</td>
			<td>BenchMate2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Micromanipulators</td>
			<td style="width:187px;">Narishige</td>
			<td>NMN-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Magnetic stands</td>
			<td style="width:187px;">ENCO</td>
			<td>Model #625-0930</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Reference Electrode Holder</td>
			<td style="width:187px;">Harvard Apparatus</td>
			<td style="width:211px;">ESP/W-F10N</td>
			<td>Can be mounted on 5ml serological pipette for extended range</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">&#160;Silver Wire</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td style="width:211px;">AGW1510</td>
			<td>0.3-0.5mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Retort Stand</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Outlet Plastic Tube</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>generic, 1cm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Flexible Plastic Tubing</td>
			<td style="width:187px;">Nalgene&#160;</td>
			<td>8000-0060</td>
			<td style="width:219px;">VI grade 1/4 in internal diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">500 ml Conical Flask</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>generic,&#160; with side arm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Aquarium Pump</td>
			<td style="width:187px;">Aquatic Gardens</td>
			<td style="width:211px;">Airpump 2000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Fiber Optic Light Source</td>
			<td style="width:187px;">Dolan-Jenner Industries</td>
			<td>Fiber-Lite 2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">White Card/Paper</td>
			<td style="width:187px;">Whatman</td>
			<td style="width:211px;">1001-110</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Digital Acquisition System</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">IDAC-4</td>
			<td>Alternative: National Instruments NI-6251 &#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Headstage</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">DTP-1</td>
			<td>Tasteprobe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Tasteprobe Amplifier</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">DTP-1</td>
			<td>Tasteprobe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Alligator Clips</td>
			<td style="width:187px;">Grainger</td>
			<td style="width:211px;">1XWN7</td>
			<td>Any brand is fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Insulated Electrical Wire</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Gold Connector Pins</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td align="right" style="width:211px;">5482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Personal Computer</td>
			<td style="width:187px;">Dell&#160;</td>
			<td style="width:211px;">Vostro</td>
			<td>Check for compatibility with digital acquisition system and software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Acquisition Software</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">Autospike</td>
			<td>Autospike works with IDAC-4; alternatively, use Labview with NI-6251</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Aluminum Foil and/or Faraday Cage</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Electro-magnetic noise shielding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Borosilicate Glass Capillaries</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td style="width:211px;">1B100F-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Pipette Puller</td>
			<td style="width:187px;">Sutter Instrument Company</td>
			<td style="width:211px;">Model P-87 Flaming/Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Beadle and Ephrussi Ringer Solution</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>See recipe in protocol section</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Tricholine citrate, 65%&#160;</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:211px;">T0252-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Stereo Microscope</td>
			<td style="width:187px;">Olympus</td>
			<td style="width:211px;">VMZ 1x-4x</td>
			<td style="width:219px;">Capable of 10x-40x magnification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Ice Bucket</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">p200 Pipette Tips</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Spinal Needle</td>
			<td style="width:187px;">Terumo</td>
			<td style="width:211px;">SN*2590</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">1ml Syringe</td>
			<td style="width:187px;">Beckton-Dickenson</td>
			<td align="right" style="width:211px;">301025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Fly Aspirator</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Assembled from P1000 pipette tips, flexible plastic tubing, and mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Modeling Clay</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Forceps</td>
			<td style="width:187px;">Fine Science Tools By Dumont</td>
			<td style="width:211px;">11252-00</td>
			<td>#5SF (super-fine tips)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">10ml Syringe&#160;</td>
			<td style="width:187px;">Beckton-Dickinson</td>
			<td align="right" style="width:211px;">301029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Plastic Tubing</td>
			<td style="width:187px;">Tygon</td>
			<td style="width:211px;">R-3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiological recording from drosophila labellar taste sensilla

استجابة طعم الطرفية من الحشرات يمكن التحقيق بقوة مع تقنيات الكهربية. الطريقة الموضحة هنا يسمح الباحث لقياس الاستجابات الذوقية مباشرة وكميا، مما يعكس المدخلات الحسية أن الجهاز العصبي للحشرات تتلقاها من المحفزات طعم في بيئته. يحدد هذا البروتوكول جميع الخطوات الرئيسية في أداء هذه التقنية. الخطوات الحاسمة في تجميع وتلاعب الكهربية، مثل اختيار المعدات اللازمة وبيئة مناسبة للتسجيل، ويتم رسم. نحن تصف أيضا كيفية الاستعداد لتسجيل بجعل المرجعية وتسجيل الأقطاب المناسبة، والحلول منبهة للذوق. نحن تصف بالتفصيل الطريقة المستخدمة لإعداد الحشرات عن طريق ادراج الإلكترود المرجعي الزجاج في ذبابة من أجل شل خرطوم. نظهر آثار النبضات الكهربائية التي أطلقها الخلايا العصبية الذوق ردا على السكر ومجمع المر. جوانب البروتوكول هي رechnically صعبة ونحن وتشمل وصفا واسعة من بعض التحديات التقنية المشتركة التي يمكن مواجهتها، مثل عدم وجود إشارة أو الضوضاء المفرطة في النظام، والحلول المحتملة. تقنية له حدود، مثل عدم القدرة على تقديم المحفزات المعقدة زمنيا، ومراقبة خلفية إطلاق النار مباشرة قبل التحفيز التسليم، أو استخدام مركبات غير قابلة للذوبان في الماء طعم مريح. على الرغم من هذه القيود، هذه التقنية (بما في ذلك الاختلافات الطفيفة المشار إليها في البروتوكول) هو المعيار، والإجراءات المقبولة على نطاق واسع لتسجيل الاستجابات العصبية ذبابة الفاكهة لتذوق المركبات.

ويبين الشكل 5A الاستجابة لL sensillum وإلى السكر، السكروز. لا يستجيب نفس sensillum والى مجمع المر، بربارين. الشكل 5B يظهر أن sensillum والنوع الأول، والذي يحتوي على الخلايا العصبية تستجيب المر، ويعرض المسامير السعة أكبر في استجابة لبربارين، وارتفاع السعة أصغر ردا...

Watch this Scientific Journal Video about Electrophysiological Recording From Drosophila Labellar Taste Sensilla at JoVE.com

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

يصف هذا البروتوكول تسجيل خارج الخلية من العمل الاستجابات المحتملة التي أطلقتها الخلايا العصبية الذوق labellar في ذبابة الفاكهة.

تسجيل الكهربية من<em&gt; ذبابة الفاكهة</em&gt; Labellar طعم الشعيرات

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

electrophysiological-recording-from-drosophila-labellar-taste-sensilla

Research

JoVE Journal

Neuroscience

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

16.2K Views.  Yale University.  يصف هذا البروتوكول تسجيل خارج الخلية من العمل الاستجابات المحتملة التي أطلقتها الخلايا العصبية الذوق labellar في ذبابة الفاكهة. 

Video: Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

في وقت واحد من تسجيل إشارات الكالسيوم من الخلايا العصبية التي تم تحديدها وسلوك التغذية من ذبابة الفاكهة السوداء البطن</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

الدماغ الأمامي تسجيل الكهربية في الزرد اليرقات

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

في وقت واحد قبل وبعد متشابك تسجيل الكهربية من<em&gt; القيطم</em&gt; العصبي العضلي المشارك الثقافات

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

غشاء المحتملة صبغ التصوير من بطني إنسي المهاد الخلايا العصبية من الفئران الكبار لدراسة الجلوكوز الاستشعار

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

تسجيل الكهربية في الدماغ سليمة من اسماك الزرد الكبار

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

وهناك طريقة لمنهجية التقييم الكهروكيميائية والكهربية العصبية من أقطاب تسجيل

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

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 ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

طعم الفحص تفضيل الكبار<em&gt; ذبابة الفاكهة</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

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.