Biz Life fonu, Oklahoma Üniversitesi Lisans Eğitim ve Araştırma Olanakları Programı, ve bu işi finanse etmek için Zooloji Bölümü teşekkür ederim. Ayrıca Dr. Marielle Hoefnagels editoryal yardım için teşekkür ederim.

1. Ön-kayıt, Canlı Hayvan Hazırlığı <ol><li> Kendi ev kavanoz akrep çıkarın ve önceden soğutulmuş bir cam kaba koyun; yanında, donmuş bir ortamda (-5 ° C) en az iki dakika akrep içeren kavanoza yerleştirin. Bu sefer yaşına göre değişebilir, boyut, çalışmanın altında özel akrep türleri, vb. Ama genelde hayvan hareketsiz bir kez, donma çevre aşaması tamamlandıktan. </li><li> İki dakika sonra, soğuk cam kap immobilize akrep kaldırmak ve bir mikroskop lamı üzerine ventral tarafta bir yerin. Stinger, kuyruk, bacak, ve moldable kil (yağ bazlı, Van Aken Plastalina) pedipalps sabitleyin. </li><li> Akrep pektinler koymak için bir platform olun. Platformu uzunluk ve genişlik Pecten boyutuna göre ayarlayın. Tipik olarak, bizim platformları mikroskop kapak camdan inşa edilmiş ve yaklaşık 10 (L) X 18 (W) mm. </li><li> Sığacak şekilde (gerekirse) bant kırparak Pecten platformu için çift taraflı yapışkan bant uyun. </li><li> Pectinal odasına yapmak için, önceden eritilmiş balmumu içine (% 100 balmumu yongaları kullanan), 3-4 mm derinliğe kadar platformun dört tarafı üç koymak. Biz sıcak bir plaka üzerinde bulunan bir cam petri içinde çipler yerleştirerek balmumu eritebilir. Pectinal odasının kenarlarında balmumu oda sıcaklığına kadar soğutulur sonra, bu yüksek yaklaşık 1 mm balmumu duvarları oluşturmanız gerekir. </li><li> Hayvan, pectinal ekleme noktasını bulun ve sadece arka platform un-mumlu kenarı (bant yan-up) yerleştirin. Daha sonra, hayvanın bacakları dizginlemek için kullanılan kil karşı sağ ve sol periferik kenarları hafifçe basarak platformu (daha sonra üzerinde hareket olmaz böylece) stabilize. </li><li> Odanın içinde Pecten veya pektinler dikkatli bir şekilde yerleştirmek için ince uçlu forseps kullanın. Özellikle, forseps kullanarak, pectinal omurga boyunca Pecten tutun ve yavaşça Pecten cam kapak altından çıkarmak ve yapışkan bir platform zemine daha düşük. Pectinal omurga dikkatlice basınç uygulayarak bir Pecten-kaset bağının kurulması. </li><li> Şimdi, platformun un-mumlu kenarına erimiş mum (önceden ısıtılmış bir metal spatula kullanın) geçerlidir. Pecten amacı güvenli ve tam geleceğin madeni yağ tanıtımı için oda içine iki yönlüdür. Çok sıcak balmumu ile Pecten zarar mümkün olduğu gibi, hayvanın Pecten üzerinde balmumu uygularken dikkatli kullanın. </li><li> Pecten odasının inşaat tamamlandı. Sonra, iki kuyruk segmentleri arasındaki gümüş tel takarak hayvan hemolimf kayıtsız bir elektrot bağlantı kurmak. </li></ol> 2. Eşzamanlı Ekstrasellüler Kayıt ve kimyasal stimülasyon <ol><li> Biz diğer nedenleri arasında (yani sabit bir pozisyonda hayvan ayarı), izin verir, ekstrasellüler, ucu kayıtları yapmak için hayvan 24 saat önce hazırlamak için zaman özel belirli bir (bağımlı sensillum) tip-çapı uyarıcı pipetler. Biz bu tür özellikleri elde etmek için bir mikropipet çektirmesi kullanın. İdeal olarak, pipetler çapı ≈ 2 mikron sensillar gözenek çapı daha büyüktür. Biz her bir hayvan için mikropipet özellikleri buna göre ayarlayın. </li><li> Kayıttan önce 1 saat, biz, petrol altında Pecten daldırarak pectinal odasına 5 ul mineral yağ ekleyin. Sonra, uzun mesafe hedefleri ve epi-aydınlatma çalışan bir yüksek güçlü mikroskop altında hayvan yerleştirin. </li><li> Kimyasal dübel sensilla teşvik etmek, sulu tastants (örneğin sitrik asit ve tuz) petrol aracılığıyla tanıtmak. İlk olarak, uyarıcı, uyarıcı pipet içine enjekte etmek için bir mikropipet dolgu kullanın. Not: Tüm uyarıcı çözümler elektriksel iletkenlik için bir elektrolit içermelidir. </li><li> Sonra, bir micromanipulator (kayıt teçhizat) uyarıcı pipet konumlandırma sonra kayıt elektrot uyarıcı pipet künt, açık ucunu yerleştirin. Metal elektrot arasındaki temas ve elektrolitik uyarıcı bir çözüm sağlamak için önemlidir. </li><li> Hazırlık elektriksel petrol aracılığıyla ve bir sensillar gözenek üzerine pipet, sadece düşük (kayıtsız elektrot aracılığıyla) topraklanmış. Pectinal sensillum başına kimyasal stimülasyon süresi kadar otuz dakika gibi kısa bir sürede ikinci değişebilir. Genellikle, pipet başına birden fazla mandalları (&gt; 30) tadabilirsiniz. Uyarıcı pipet dakika enkaz ile tıkanmış hale gelmesi durumunda, yeni, kullanılmamış bir pipet tıkanmış pipet değişimi. </li></ol> 3. Hayvan Deneyleri Çoklu Günler için hazırlanması <ol><li> Hayvan uzun bir süre boyunca kullanılmak üzere ise, pectinal odasının yeniden ve her kayıt oturumu için mineral yağ damla yerine öneririz. , Belirli bir günde kayıt işlemi tamamlandıktan sonra, kayıt set-up hayvan çıkarın ve bir madeni yağ yıkama yapın. </li><li> Specifically, mineral yağ, başka bir odaya açılan (5 ul) ekleyin ve tüm petrol çıkarmak. Bu kayıt oturumu sırasında pipetin dışarı sızdırılmış olabilir kalıntı uyarıcı, varlığı en aza indirmeye yardımcı olur. </li><li> Sonraki oturumda önce, daha önce açıklandığı gibi bir damla yağ ekleyebilirsiniz. </li></ol> 4. Temsilcisi Sonuçlar: N oranı en az 3:1 kayıt ekipmanları (özellikle amplifikatör gibi, sayısallaştırma donanım, vb.) Bağlı olarak, temsili bir sonuç S ile ekstraselüler kayıt. Başarılı yapım, mineral yağ haznesi, kimyasal uyarımı bireysel sensilla izole için gerekli olan polar (su bazlı) kimyasal tastants 8 sağlamak için bir ortam sağlar . Çok az yağ ya da hiç yoksa, uyarıcı bir çözüm, uzun bir kimyasal stimülasyon 8, kontrolsüz bir maçı neden olabilecek komşu sensilla kaydedilen sensillum yayılacaktır.

<ol>
	<li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li><li>Carthy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li><li>Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li><li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li><li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li></ol>

Biz daha önce iki dergi, Knowlton ve Gaffin (2009) ve Knowlton ve Gaffin (baskıda) bu yöntemlerin yayınlandı.

Yukarıdaki protokol elektrofizyolojik çalışma için bir çöl otlak akrep (Paruroctonus utahensis) nasıl hazırlanacağını açıklar. Özellikle, biz altında pectinal yağ kimyasal-duyusal nöronların kontrollü ekstrasellüler ucu kayıtları için nasıl bir oda inşa göstermektedir. Yağ ve su karışmaz, çünkü kimyasal stimülasyon (su bazlı uyarıcılar ile) tek sensilla izole etmek mümkündür. Vurgulamak gerekir ki, P. utahensis, görece küçük bir hayvan (≈ 2.5-5 cm) ve büyük hayvanların bu protokolü uygulayarak hayvanın platformu, odanın boyutu gibi birçok boyutu ayarlamaları ve uygulanan yağ miktarı gerekebilir. Biz su bazlı kimyasal uyarıcıların kontrollü teslimat için gerekli minimum miktarda yağ test etmek için pilot çalışmalar yapmanızı öneririz. Ayrıca, yağ içinde pectinal sensilla 8 temel sinir aktivitesi üzerinde herhangi bir etkisi bulundu. Bu önce chemoresponsiveness değerlendirirken, aynı zamanda diğer model sistemler için teyit edilmelidir. Bu yöntem chemoresponse duyusal alanında sensilla arasında etkileşim etkileri test etmek için başka bir yöntem ile birlikte kullanılabilir. Örneğin, bir sensillum ucu-record (yağ altında) mümkün olduğu gibi biz komşu bir sensillum 9 baz kayıt (tungsten elektrod aracılığıyla). Böyle bir kayıt yapılandırma tabanına kaydedilmiş sensillum nöral aktivitenin etkiler olsun ya da kimyasal bir sensillum uyarıcı değerlendirmek için kullanılıyor olabilir. Bugüne kadar hiç kimse bu akrep pectinal sensilla test etmiş ve bunun yanı sıra diğer kimyasal-duyusal sistemleri için açık bir soru kalır. Özetlemek gerekirse, biz, petrol gelişmeler eklembacaklı tadında nöral temeli haline elektrofizyolojik araştırmalar kapsamı altında ucu-kayıt yöntemi düşünüyorum. Bu yazıda, biz bu yöntem için hayvan hazırlamak için bir protokol sağlamak ve periferik duyusal sinir sistemi daha fazla çalışma için sağlam bir temel sağlar umuyoruz.

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<td>Lift-N-Press tab</td>
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electrophysiology of scorpion peg sensilla

Biz, kısa, duyusal sensilla 3,4 tahta gibi elektrofizyolojik araştırmak için, mevcut bir ucu kayıt tekniği 1,2 bir değişiklik açıklar. Bütün akrepler orta-ventral yüzey üzerinde yoğun alanlar var pektinler olarak adlandırılan iki ekleri, mekanik ve kimyasal-duyusal peg sensilla 5,6 . Bu sensilla chemoresponsiveness değerlendirilmesi için bir yöntem uçucu odorant duyusal alanında 5,7 olarak ekstraselüler sensillum içinde nöral aktiviteyi kayıt için bir tungsten elektrot kullanır. Bu yöntemin sınırlarını yavaş veri toplama ve kontrolsüz uyarıcı giriş ve Hazır Giyim alanında, kaldırılması içerir. Bu sınırlamaları aşmak için, biz, bireysel peg sensilla 8,9 su bazlı tastants sunmak için bir araç olarak apolar madeni yağ kullanan yeni bir ucu kayıt tekniği geliştirdi . Biz başarıyla sensillar chemoresponses, sitrik asit, etanol, ve tuz elde etmek için bu yöntemi uyguladık. Burada, böyle bir çalışma 9 için deney protokolünde açıklar . Biz bu yeni yöntem, bu böcekler 10, 11 ve 12 kabuklular de dahil olmak üzere diğer eklembacaklı kimyasal-duyusal sistemleri, tepki özelliklerini inceleyerek için yararlı olabilir düşünüyorum .

Watch this Scientific Journal Video about Electrophysiology of Scorpion Peg Sensilla at JoVE.com

Electrophysiology of Scorpion Peg Sensilla

Bu makale, mineral yağ altında ekstrasellüler, ucu kayıtları üzerinden bireysel sensilla kimyasal stimülasyon izole bir elektrofizyolojik yöntem açıklanır.

Akrep Peg Sensilla elektrofizyolojisi

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

electrophysiology-of-scorpion-peg-sensilla

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.0K Görüntüleme.  University of Oklahoma. Bu makale, mineral yağ altında ekstrasellüler, ucu kayıtları üzerinden bireysel sensilla kimyasal stimülasyon izole bir elektrofizyolojik yöntem açıklanır.

Video: Electrophysiology of Scorpion Peg Sensilla

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

Kemirgen Beyin aynı anda fMRI ve Elektrofizyoloji

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Nörobilim

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Omurilik Elektrofizyoloji II: Ekstrasellüler Emiş Elektrot İmalatı

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Hücreler NRCC Patch-klemp Chips kültür ve Elektrofizyoloji

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Kullanarak Bileşiklerin Biyoaktivite için eşli Nanoinjection ve Ekran için Elektrofizyoloji Testi Drosophila melanogaster Dev Elyaf Sistemi

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Elektrofizyoloji ve yüksek çözünürlüklü İki foton Mikroskopi için Fare Vestibüler Duyu epiteli bir İzole Yarı sağlam Hazırlık

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Elektrofizyolojik Kayıt<em&gt; Drosophila</em&gt; Labellar Sensilla Taste

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

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Gerçek zamanlı Elektrofizyoloji: Probe Nöronal Dynamics ve Ötesi Kapalı çevrim Protokolleri Kullanma

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Yenidoğan Kemirgenler İzole Beyin Sapı-spinal Kordon Müstahzarlar Elektrofizyoloji Sinir Solunum Ağı Çıktı Kayıt verir

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Çok elektrot dizilerde Spiral Ganglion Nöron Eksplantasyon Kültür ve Elektrofizyoloji

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Elektrofizyoloji ve Kalsiyum-görüntüleme için akut nöronal Doku Uzamış İnkübasyon

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...