Bu iletişim kuralı araştırma hibe Ulusal Doğa Bilimleri Foundation of China (#31171066, #81270464) ve Sino-Alman bilim merkezi (GZ919) tarafından desteklenmiştir.

hayvan Etik Komitesi, Shanghai Jiaotong Üniversitesi Tıp Fakültesi tarafından (# SYXK2013-0050) rapor burada deneysel protokol onaylandı. sağlam ganglion ve sinir colorectum diseksiyon gövde Bu teknikte oldukça deneyimli bir kişi için en az 15 dakika alır. Bu nedenle sonraki Elektrofizyolojik kayıt için dokuların canlılığı sağlamak için gruptakiler, performans iken hayvan hayatta ama derin anestezi altında tutmak gereklidir.
 1. hazırlık perfüzyon çözüm ve Test bileşikleri <ol><li>hazırlamak 5 L Krebs çözüm: 113 mM NaCl, 5.9 mM KCl, 1,2 mM NaH 2 PO 4, 1,2 mM MgSO 4, 25 mM NaHCO 3, 1,2 mM CaCl 2 ve 11.5 mM glikoz. Çözüm emdirmek % 95'i O 2 + % 5 CO 2 gaz karışımı ile. Önceden buzdolabından oksijenli Krebs ~ 500 mL serin.</li>
	<li>Hazırla aliquots stokunun gerektiği gibi bileşikler (etanol ve 10 mM 5-hydroxytryptamine (5-HT) serum fizyolojik içinde 1 mM kapsaisin) sınayın. Hisse senedi Krebs (için banyo uygulaması) ve serum (intraluminal Yönetim için) son toplama kullanmak için sadece önceki sulandırmak.</li>
</ol> 2. Kayıt elektrodu hazırlanması <ol><li>kayıt elektrot çift cam tüpler bir geleneksel elektrot çektirmenin kullanarak iç filamentler (1.5 mm dış çap) çekme. Isı çektirme ayarlarını ve böylece bıçak çekti elektrot arasında 20 çekin ve 25 mm.</li>
</ol> 3. Doku koleksiyonu <ol><li>uyuşturan derinden sodyum Fentobarbital (80 mg/kg, IP) kullanarak fare. </li> <li>Kısırlık sağlarken, karın boşluğu bir neşter kullanarak karın duvarına bir ensizyon uygulayarak maruz. Bıçaklanmasından ve colorectum ortaya çıkarmak için diğer dokulara bir kenara çekin.</li>
	<li>Hayvan diseksiyon mikroskop altında yerleştirin. Dikkatli diseksiyon, sol PG bulun ve PN ve katılarak SSN tanımlar. Bu sinirler sayfa uzak birkaç milimetre kesme 
	Not: PG kolorektal Kavşağı yakınında yer almaktadır. Genellikle, Lumbosakral spinal kord ve SSN projesinde 3-4 PN için PG ( şekil 1).</li>
	<li>Rektum ortaya çıkarmak için symphysis kemik kesme. Colorectum yukarıda belgili tanımlık doku (Yani, idrar kesesi, vb) kaldırın; PG olduğu gibi bırakmak için dikkatli olun.</li>
	<li>Sıçan Fentobarbital aşırı dozda intravenöz enjeksiyon tarafından kurban. İki nokta üst üste yaklaşık 3 cm yukarıda PG transect ve forseps kullanarak hayvandan colorectum kaldırmak.</li>
	<li>Colorectum önceden soğutulmuş Krebs çözüm ile dolu bir Petri kabına aktarın. Belgili tanımlık feces yavaşça iki nokta üst üste temizlenerek kaldırın. Kalan idrar kesesi ve diğer dokuların dikkatli bir şekilde, sayfa ödün vermeden çıkarın</li>
</ol> 4. Diseksiyon kolon Afferent sinir <ol><li>iki nokta üst üste kayıt odası (20 mL) yerleştirin ve doku carbogenated Krebs çözüm ile sürekli sıvı. Set perfüzyon oranı 15 mL/dak.</li>
	<li>Her iki ucunda oral ve anal colorectum cannulate. 10 mL/h oral anal yönüne oranında salin ile iki nokta üst üste intraluminal infüzyonu başlatın.</li>
	<li>Büyük PG diseksiyon mikroskop altında bulun. Böcek pimleri ganglion ortaya çıkarmak için kullanın. Dikkatli diseksiyon ile sinir gangliyon yayılan ve iki nokta üst üste doğru ( şekil 1) çalışan iyi bir dalı bulmak. Sinir gangliyon yakın kesti. 
	Not: Resim 1 PG distal bir sinir dallara bir kayıt gösterir. Sinir muhtemelen pelvik ve Lomber splanchnic afferent lifler karışımı içerir. Alternatif olarak, bir kayıt PN ve/veya sayfa için proksimal SSN yapılabilir</li>
	<li>Isıtma banyo ve sıcak 34 ± 0,5 ° c oda sıcaklığında tutmak için Krebs çözüm açmak</li>
</ol> 5. Emme elektrodu hazırlanması <ol><li>önceden çekti cam pipet (Adım 2) almak ve diseksiyon mikroskop altında incelemek. Kaydedilmek üzere sinir çapı ile uyumlu bir büyüklükte olduğu elektrot ucunu forseps bir çift ile break.</li>
	<li>Daha hafif bir parlama yakın yerleştirerek uç eğim.</li>
</ol> 6. Elektrofizyolojik kayıt <ol><li>Bağlan eğimli elektrot elektrot tutucu için. Yan bağlantı noktasına tutucu elektrot için negatif veya pozitif basınç uygulamak için bir 10 mL şırınga takın.</li>
	<li>Sahibi bioamplifier headstage için bağlanmak ve headstage bir manipülatör üzerine monte edin.</li>
	<li>Elektrot doku Kaplıcalar'a hareket ve elektrot sahibinin gümüş tel bağlantı kurduğunda kadar nazik emiş uygulayarak Krebs çözüm ile doldurun. Elektrot uç sinir kesme sonuna yakın koyun ve elektrot sinir emmeye negatif basınç uygulayın. Sinirin ~ 1 mm elektrot çekti ve sıkı bir mühür formları daha negatif basınç uygulamak.</li>
	<li>Dönüş bioamplifier ve küme filtre için 300-3.000 Hz. osiloskop üzerinde sinyal izlemek ve sinir sinyal (20 kHz örnekleme hızı) ve bir bilgisayar ile bir spike veri işleme kullanarak intraluminal basınç sinyal (100 Hz örnekleme hızı) kayıt yazılım.</li>
</ol> 7. Kolon Afferent duyarlılık test <ol><li>Uygula rampa distansiyonu sürekli intraluminally beslerken ise çıkış kanül üç yönlü mekândaki kapatarak kolon. Bu üç yollu musluk boşaltma kanül üstünde hangi zamanda açık 60 mmHg, ulaşıncaya kadar intraluminal basınç izlemek.</li>
	<li>15 dk. Uygula uyuşturucu ilave - veya intraluminally afferent sinirlerin kimyasal duyarlılık test etmek için düzenli aralıklarla bu yordamı yineleyin.</li>
</ol>

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Yazarlar hiçbir çıkar çatışması beyan ederim.

Burada sunulan sıçan kolon afferents Elektrofizyolojik özelliklerini değerlendirmek için nispeten basit bir deneysel yöntem protokolüdür. İletişim kuralından (doku diseksiyon) kadar sinir kayıt ayarı genellikle tamamlamak için yaklaşık 2 saat sürer. Doku toplama (Adım 3) ve emme elektrot (5. adım) hazırlanması önemli adımlardır. PG, SSN ve PN bulun ve sinirler ve sinir dokusu diseksiyon sırasında zarar vermemeye dikkat çekmek için önemlidir. Cam pipet ucu kırık ve sinir paket ile uyumlu bir...

Gastrointestinal sistem (GIT) zengin dışsal afferent sinir duyusal gut merkezi sinir sistemi için sinyalleri iletmek ve bu gut-beyin etkileşimine katkıda innervated. Bu dışsal afferents değişmiş uyarılabilirlik, hem de değiştirilmiş merkezi işlem afferent girdilerin visseral ağrı ve diğer semptomlar işlevsel ve enflamatuar barsak hastalıkları1de dahil olmak üzere GI koşullardan altında yatan. Colorectum gelen duyusal bilgileri öncelikle thoracolumbar/hypogastric ve Lumbosakral/pelvik sinirler (PN)2ile aktarılmaktadır. Kemirgen hastalık modelleri bu birincil afferent liflerinin Elektrofizyolojik özellikleri eğitim artan bir ilgi oluştu. Ancak, in vivo Elektrofizyolojik kayıtları Rodents kolon afferents teknik bir sorundur ve önemli cerrahi becerileri gerektirir. Buna ek olarak, hemodinamik değişiklikler, doku hareketi ve anestezi de sinir etkinliği ve hassasiyetini uyaranlara vivo içindetest etmek için bazı etkileri olur. Bu nedenle, son yıllarda, farklı tür, dahil olmak üzere fare, rats, eskiden şiling şimdi pigs ve insanlar, vitro (ex vivo) hazırlıkları içinde kolon duyusal iletim mekanizmaları incelemek için çalışmalar giderek artan sayıda istihdam afferents ve hastalık koşullarında değişmiş uyarılabilirlik. 3 , 4 , 5 , 6 , 7 , 8
Ex vivo kolon hazırlık iki tür öncelikle bildirilmiştir: "düz sayfalık" hazırlık5,9,10 ve "tüp" hazırlık3,4. Video iletişim kuralı "düz sayfalık" fare colorectum hazırlık için daha önce yayımlanmış11oldu. Bu protokol, fare colorectum ile PN) veya lomber splanchnic sinirler (SSN) bağlı, hasat ve superfused bir doku odasında. Colorectum boyuna ikiye ayıracağım ve sinir demeti parafin yağı ile dolu bir kayıt bölme içine genişletilir. Sinir faaliyet monopolar Platin-iridyum elektrot kullanarak kaydedilir. Protokol bireysel afferent liflerinin alıcı alanları tanımlanması için tarafsız elektriksel stimülasyon kullanarak sağlar. Kimyasal uyaranlara uygulanması, hem de uygulamanın farklı mekanik stimülasyon paradigmaların yerelleştirir (Örneğin, Fokal Mukozal sondalama ve çevresel streç), afferent sinir uçlarının için. Sinir doku odasından ayrı bir odaya genişlemelidir çünkü bağlı sinir nispeten uzun tutmak için önemlidir; sinirleri başarılı diseksiyon Bu metodoloji yeni bu bir meydan okuma teşkil. Daha yakın zamanlarda, Nullens ve ark. Mezenterik afferents in vitro kayıt için video bir protokol fare jejunal ve kolon kesimleri12yayınlandı. Bu "tüp" hazırlanmasında bağlı bıçaklanmasından gut segment bozulmamış, böylece kademeli distansiyonu ve farklı kimyasallar içi ve ekstra luminal Yönetim için izin tutulur. Bıçaklanmasından sinir doku yakın konumlandırılmış olabilir, bir emme elektrot kullanarak kaydedilen bu yana bıçaklanmasından sinir nispeten kısa olsa bile afferent etkinlik kaydedilebilir. Ancak, bıçaklanmasından sinir değişir innervate Vagus ve omurga afferent liflerinin karışık nüfus oluşur veya thoracolumbar hypogastric. Lumbosakral pelvik afferents bu protokol için ayrımcılık colorectum innervate. Burada, sıçan kolon afferents "tüp" colorectum hazırlık ile sağlam bir PG kullanarak Elektrofizyolojik kayıt için detaylı bir iletişim kuralı mevcut Bu yöntem lomber fonksiyonel özelliklerini karakterizasyonu için splanchnic izin verebilir (hypogastric) ve Lumbosakral pelvik afferents.

<table><tbody><tr><td>Sodium Pentobarbital</td><td>Shanghai Westang Bio-Tech</td><td>B558</td><td></td></tr><tr><td>Capsaicin</td><td>Sigma</td><td>M2028</td><td></td></tr><tr><td>Electrode puller</td><td>MicroData Instrument Inc</td><td>PMP107</td><td></td></tr><tr><td>Neurolog System (Bioamplifier)</td><td>Digitimer, Ltd</td><td>Neurolog System</td><td></td></tr><tr><td>A/D converter</td><td>Cambridge Electronic Design</td><td>Micro1401</td><td></td></tr><tr><td>Data processing software</td><td>Cambridge Electronic Design</td><td>Spike2 version 6</td><td></td></tr><tr><td>Silver wire</td><td>World Precision Instruments</td><td>EP12</td><td></td></tr><tr><td>Glass tubes</td><td>World Precision Instruments</td><td>1B150-4</td><td></td></tr><tr><td>Electrode holder</td><td>World Precision Instruments</td><td>MEH3SBW</td><td></td></tr><tr><td>Heating bath</td><td>Grant</td><td>GR150</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>Zoom2000</td><td></td></tr><tr><td>Dissecting microscope</td><td>World Precision Instruments</td><td>PZMIII-BS</td><td></td></tr><tr><td>Cigarette lighter</td><td>any</td><td>NA</td><td></td></tr><tr><td>Surgical tools</td><td>World Precision Instruments</td><td>NA</td><td></td></tr><tr><td>Insect pins</td><td>home-made from 0.1 mm stainless steel wire</td><td>NA</td><td></td></tr><tr><td>Three way manipulator</td><td>World Precision Instruments</td><td>KITF-R</td><td></td></tr><tr><td>Rats</td><td>Any</td><td>NA</td><td>Any strain/sex can be used.</td></tr></tbody></table>

in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats

Disfonksiyon kolon duyusal sinir işlevsel ve enflamatuar barsak hastalıkları ve diyabet gibi birçok ortak koşulları Patofizyoloji karıştığı olmuştur. Burada, kolon afferents Sıçanlarda Elektrofizyolojik özellikleri vitro karakterizasyonu için bir protokol açıklayın. Colorectum bağlı, bozulmamış pelvik gangliyon (PG) ile sıçan gelen kaldırılır; carbogenated kayıt odasında Krebs çözüm ile superfused; ve distansiyonu için izin vermek için oral ve anal ucunda cannulated. PG yayılan bir iyi sinir paket tanımlanır ve multiunit afferent sinir faaliyet emme bir elektrot kullanarak kaydedilir. Distansiyonu kolon segmentin kademeli multiunit deşarj artışlar ortaya çıkarır. Düşük eşikli, yüksek eşikli ve geniş dinamik sıra afferent lifleri ayırmak için asıl bileşen analizi yapılır. Kolon afferents kimyasal duyarlılık testi bileşikler banyo veya intraluminal yönetim okudu olabilir. Bu iletişim kuralı uygulama için diğer türler, fare ve eskiden şiling şimdi pigs gibi ve thoracolumbar/hypogastric ve Lumbosakral/pelvik afferents inen kolon normal Elektrofizyolojik özelliklerindeki farklılıklar çalışmaya değiştirilebilir ve patolojik durum.

Şekil 1 bir sinir distal PG için kayıt bir temsilci ile ex vivo "tüp" colorectum hazırlık için deneysel kurulumunun şematik çizimde olduğu Sinir muhtemelen pelvik ve bel splanchnic afferents karışımı yer. Normal sıçanlar üzerinden hazırlıkları içinde kolon afferent sinirlerin genellikle düzensiz spontan aktivite düşük düzeyde var. Rampa distansiyonu kolon ateş oranı (şekil 1B) kademeli bir ar...

Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Anormal duyusal fonksiyonu altında yatan visseral ağrı ve diğer semptomlar işlevsel ve enflamatuar barsak hastalıkları. Bir ex vivo sıçan colorectum hazırlık kolon afferent sinirlerin Elektrofizyolojik kayıt için bir protokol burada sunulmaktadır.

Vitro Sıçanlarda kolon Afferent liflerinin Elektrofizyolojik özellikleri karakterizasyonu

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

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In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

6.9K Görüntüleme.  Shanghai Jiaotong University School of Medicine. Anormal duyusal fonksiyonu altında yatan visseral ağrı ve diğer semptomlar işlevsel ve enflamatuar barsak hastalıkları. Bir ex vivo sıçan colorectum hazırlık kolon afferent sinirlerin Elektrofizyolojik kayıt için bir protokol burada sunulmaktadır.

Video: In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Sıçanlarda Sakkaroz Craving Kuluçka değerlendirilmesi için Genel Bir Yöntem

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Nörobilim

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

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

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

Bozulmamış Retina Elektrofizyolojik Karakterizasyonu GFP-ifade eden hücre popülasyonlarının

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

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

Fare Kolorektal Afferent Endings in vitro Fonksiyonel Karakterizasyonu içinde

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Tıp

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

Fare jejunal ve Kolon Segmentlerde Mezenterik Afferent Sinir Faaliyet Vitro kaydedilmesi

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Vivo Aksonal uyarılabilirlik test ile sıçan Ulnar sinirin Elektrofizyolojik ölçüm

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

İletim Yetmezliği Mekanizmasını İncelemek Için Bağlı Siyatik Sinir ile In Vivo Tek Lifli Kayıt ve Bozulmamış Dorsal Kök Ganglion Kullanımı

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Electrophysiological Recording from Colonic Afferent Nerves in an Ex Vivo Rat Colon

Source: Meng, Y. et al., <a href="https://app.jove.com/v/56090">In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.</a> J. Vis. Exp. (2017)
This video demonstrates a method to record the activity of colonic afferent nerves in an ex-vivo rat colon, cannulated at both ends and infused with saline to increase intraluminal pressure. As pressure increases, the nerve signal rises, indicating its stimulation.

Bir Ex Vivo Sıçan Kolonunda Kolon Afferent Sinirlerinden Elektrofizyolojik Kayıt

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

In Vivo Single-Fiber Recording of Sciatic Nerve C-Fibers in Rats

Source: Mao, H., et al. <a href="https://app.jove.com/v/59234">Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure.</a> J. Vis. Exp. (2019).
This video demonstrates a single-fiber recording of C-fiber nerve conduction in rats. An anesthetized rat is incised to expose the sciatic nerve, which provides sensory and motor supply to the lower limbs. The individual C-fibers are then exposed. Electrical stimulation is provided using stimulus electrodes implanted in the foot, and a response is recorded from a single nerve fiber.

Sıçanlarda siyatik sinir C-liflerinin in vivo tek lif kaydı

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

In this procedure, using a micromanipulator, lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand, and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath.
To verify the recording of afferent nerve activity, perform a ramp distension-induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge.