Name: electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording
Uploaded: 2015-11-19T13:00:00.000+00:00
Duration: 5 min 28 s
Description: Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

Bu yöntem, Laval Üniversitesi Hayvan Etik Komitesi (protokol # 2012-170) tarafından izin verilen hayvan deneklerin kullanımını gerekli. 1. Kurulum ve Hazırlık <ol><li> Çözümler <ol><li> Aşağıdaki tarifleri 7,17 göre aCSF stok çözümleri hazırlayın. Konsantrasyon varyasyonları ile diğer yemek tarifleri literatürde mevcuttur. En fazla bir ay süreyle 4 ° C&#39;de saklayın stok çözeltileri. <ol><li> Tuz çözüm: NaCl 75,39 g (129 mM nihai) ekleyin; KCl 2,5 g (3,35 mM nihai); NaH2PO4, 0.81 g (0.58 mM nihai); MgCl2 2.33 g (1.15 mM nihai); CaCl2 1.85 g (1.26 mM). Daha sonra damıtılmış su ile 1 L damıtılmış su içinde dolgu 800 ml içinde çözülür. </li><li> Bikarbonat çözeltisi: NaHCO 3 (21 mM nihai) 17.65 g ekleyin. Daha sonra damıtılmış su ile 1 L damıtılmış su doldurun 900 ml içinde çözülür. Bikarbonat konsantrasyonunun varyasyonlar pH varyasyonları ile sonuçlanacaktır. </li><li> Glikoz çözüm: 54.06 ekleyinglikoz g (30 mM nihai). Daha sonra damıtılmış su ile 500 ml&#39;ye kadar damıtılmış su doldurun 400 ml içinde çözülür. </li></ol></li><li> Tuz çözeltisi 100 ml, bikarbonat çözeltisi, 100 ml damıtılmış su, 1 L glikoz solüsyonu 50 ml seyreltilerek aCSF hazırlayın. Kullanılana kadar 4 ° C&#39;de saklayın. </li><li> Isınma ve kopana kadar bir cam tüp gererek cam elektrot hazırlayın. Kullanmadan önce ucu aşağı Kum. Elektrot sürece temiz kalır gibi birçok zaman yeniden kullanılabilir. </li></ol></li><li> Deneysel kurulum Not: kurulum detayları Şekil 2&#39;de sunulmuştur. <ol><li> Ortalaması alınmış, veri toplama sistemi, sıcaklık kontrolörü ve pompayı hareketli, amplifikatörü açın. Ve ham sinyal (2.5 kHz) dijital dönüşüm analog örnekleme oranını, sinyal amplifikasyon (= 10,000 kazanç), filtrasyon (yüksek eşik, 5 kHz düşük eşik, 10 Hz) kontrol edin. </li><li> ACSF ve karbojen (% 95 O 2 ile kabarcık onu bir şişe doldurun </sub&gt;% 5 CO2). / Dakika 4 ml&#39;de kayıt odasına (hacim 5 mL) &#39;de bir bubbled- ve ısıtılmış aCSF akış neden. ° 26 de kayıt bölümü içinde sıcaklığı (± 1) tutun. pH, bu standart koşullarda 7,4 olmalıdır. </li><li> Diseksiyon odasının yakınında, bir 50 ml şırınga içinde, oda sıcaklığında ve karbojen-kabarmış aCSF 50 ml tutun. </li><li> Bilgisayarı açın ve kayıt yazılımı başlatın. </li></ol></li></ol> 2. Diseksiyon <ol><li> Tartılır ve görsel hayvanın cinsiyeti belirlemek (kadın kısa ano-genital mesafe varken erkek, uzun bir ano-genital mesafe var; kadın genital pembe iken genital genellikle koyu erkeklerin). </li><li> Aşağıdaki seçeneklerden birine göre yeni doğmuş kemirgenler anestezisi: cryoanesthesia (tam 4 buz içinde hayvan daldırın - 5 dakika 18) (4 ml / kg equitensine), enjeksiyon, uçucu anestezikler (izofluran 20 veya eter 21) 19 ya da inhalasyon. Confirm Bir pençe geri çekme refleksinin yokluğu ile anestezi yeterli bir uçak. </li><li> Aşağı bankta hayvan, ventral yüzü koyun. Bölüm (deri ve kafatası yoluyla görülebilir) Bregma düzeyinde bir neşter ile koronal başın rostral parçası. Hayvan anestezi hemen sonra bu adımı gerçekleştirin. </li><li> Bölüm vücut koronal anterior üyeleri altında bir neşter ile. </li><li> Cerrahi ve ince makas ve pense ile cilt, kaslar, yağ doku ve organ çıkarın. Kemikler bu yaşta yumuşak olduklarından, sinir sistemine zarar değil dikkatli olun. Bankta bu adımı gerçekleştirin. </li><li> Diseksiyon odasında hazırlık yerleştirin. Preparasyonunu oksijen şırınga saklanan aCSF kullanın. Kaydın sonuna Bu açıdan bakıldığında, bir mikroskop kullanılır. </li><li> Hazırlık dorsal yüzünde, cerrahi ve ince makas ve pense ile orta eksen boyunca kaudal kısmına rostral gelen kafatası ve vertebralar kesti. Kesim kafatası açınve sırayla omurganın sinir dokusunun ortaya çıkarmak. Preparasyonunu oksijen olması enjektördeki strored aCSF kullanın. </li><li> Pense ile Araknoid membran, sinir dokusunun yüzeyini örten ince bir doku çıkarın. Sinir dokusunun karşı pia mater ve kan damarlarını korur. Preparasyonunu oksijen şırınga saklanan aCSF kullanın. </li><li> Dorsal ile hazırlık yüzü aşağı gelecek şekilde ve dikkatle yerine hazırlık tutarken makasla sinirleri ve bağ dokusu kesilerek beyin sapı ve omurilik aşağı getirmek yerleştirin. Bir dorsal yaklaşım da mümkündür. Mümkün olduğunca uzun kökleri ve sinirleri tutun. Beyin sapı ve omurilik izole etmek için kemikleri çıkarın. Preparasyonunu oksijen şırınga saklanan aCSF kullanın. </li><li> Beyincik çıkarın ve neşter ile kesit ile rostral yapıları kalan. Preparasyonunu oksijen iğne saklanan aCSF kullanın. </li><li> İsteğe bağlı olarak experimen bağlı olaraktal tasarım, alt serebellar arterin 22 koronal kesit ön tarafından neşterle pons kaldırın. Bütün pons tutarak sıçanlarda ritmi yavaşlatmak ve tam farelerde bunu inhibe unutmayın. Pons sadece kaudal bölümünü içerir hazırlıklar C4 kökünde istikrarlı bir frekans ile solunum benzeri aktivite gösterirler. </li></ol> 3. Kayıt <ol><li> Kayıt odası, ventral yüzü yukarı hazırlık yerleştirin. Omurilik ve beyin sapında rostral-çoğunlukla en alt kısmında yer pimleri ile hazırlık sabitleyin. </li><li> Kısmen aCSF ile elektrot doldurmak için, şırınga pistonu çekerek: - (225 mikron 150 cam ucu çapı) olan iğne ile elektrot bağlanan bir şırınga kullanarak, elektrot bir depresyon neden olur. </li><li> Bir mikromanipülatör kullanarak, dikkatle dördüncü ventral köklerin birine yakın elektrot yerleştirin. Diğer ventral kökleri de solunum benzeri aktivite (e sunabilir.g., XII kranial sinir, C1 ventral kök). </li><li> Yavaşça sinir kökçük aspire için pistonu çekerek şırıngayla elektrot tankında bir depresyon neden olur. Sonra dikkatlice omurilik karşı uygulamak için elektrot taşıyın. Not: İdeal olarak ince kök boyutu elektrot açıklığının boyutu ile eşleşen ve böylece elektrot iç ve dış bölümler arasında bir sızdırmazlık sağlar. Diferansiyel amplifikatör genellikle bu tür kayıtlar için kullanılan bu yana, elektrodun iç ve kayıt odasının arasında herhangi bir açıklık sinyalinin kalitesini düşürür ve zor arka plan gürültüsünü ortadan kaldırmak için yapar. </li><li> Kayda başlayın. </li><li> Normoksik koşullar altında hazırlanması tarafından üretilen ritmi kaydedin (örneğin, aCSF karbojen ile fokurdatıldı:% 95 O2 ve% 5 CO2), en az 20 dakika Preparat başlangıç ​​parametrelerini belirlemek için. </li><li> Için karbojen-kabarmış aCSF gelen perfüzyon Anahtarıuyaran CSF (yani,% 95 N2 ve% 5 hipoksi uyarıcı CO2 ile kabarcıklandırılmış) 15 dakika karıştırıldı. Deneysel protokol bağlı pozlama süresini değiştirin. Kayıt ve hayvan veri sayfasında pozlama süresini kaydedin. ACSF şişe ve tüp dışına gaz difüzyonu önlemek için mümkün olduğunca odasına arasındaki paslanmaz çelik borular kullanın. </li><li> Kurtarma kayıt için en az 15 dakika süreyle standart karbojen-kabarmış aCSF geri perfüzyon geçin. Kayıt ve hayvan veri sayfasında bu kaydedin. </li><li> Kaydı bitirmek. </li></ol> 4. İstatistiksel Analiz <ol><li> Entegre sinyal, (patlamaları / dak olarak ifade edilmiştir) Dakikada patlamaları sayısı sıklığı, başlangıç ​​ve (mV) olarak ifade patlama tepe arasındaki fark olarak genlik gelen süresi olarak patlama süresini hesaplamak (s) olarak ifade patlama sonuna başlangıcı, cur altındaki alan olarak patlama alanı(mV cinsinden ifade edilmiştir · s), entegre bir sinyal (Şekil 3) kaymasının ettik. </li><li> Her durum için kayıtların son 5 dakika ortalama olarak frekans, genlik, patlama süresi ve patlama alanı normoksik altında (başlangıç), hipoksik (uyaran) ve post-hipoksik (post-uyaran) kurtarma koşullarını hesaplayın. Kayıt odasına perfüzyon ve aCSF homojenizasyon anahtarlama arasında bir gecikme olduğundan, her durumda ilk kısmını analiz etmeyin. </li><li> İlgili kayıt için temel değerlerinin bir yüzdesi olarak daha sonra uyaran (örneğin, hipoksi) ve post-uyaran (örneğin, post-hipoksik) kurtarma değerleri ifade eder. SD ± aracı olarak sonuçları Express </li></ol>

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S., Denavit-Saubie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+NMDA+receptors+in+the+respiratory+phase+transition+is+different+in+the+adult+guinea+pig+in+vivo+and+in+the+isolated+brain+stem+preparation.">Involvement of NMDA receptors in the respiratory phase transition is different in the adult guinea pig in vivo and in the isolated brain stem preparation.</a> J Neurophysiol. 74, 770-778 (1995).</li><li>Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+volatile+anesthetics+on+respiratory+activity+and+chemosensitivity+in+the+isolated+brainstem-spinal+cord+of+the+newborn+rat.">Effects of volatile anesthetics on respiratory activity and chemosensitivity in the isolated brainstem-spinal cord of the newborn rat.</a> Hokkaido Igaku Zasshi. 73, 117-136 (1998).</li><li>Gestreau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Task2+potassium+channels+set+central+respiratory+CO2+and+O2+sensitivity.">Task2 potassium channels set central respiratory CO2 and O2 sensitivity.</a> PNAS. 107, 2325-2330 (2010).</li><li>Caravagna, C., Soliz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+and+MEK+molecular+pathways+are+involved+in+the+erythropoietin-mediated+regulation+of+the+central+respiratory+command.">PI3K and MEK molecular pathways are involved in the erythropoietin-mediated regulation of the central respiratory command.</a> Respir Physiol Neurobiol. 206C, 36-40 (2014).</li><li>Tree, K., Caravagna, C., Hilaire, G., Peyronnet, J., Cayetanot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anandamide+centrally+depresses+the+respiratory+rhythm+generator+of+neonatal+mice.">Anandamide centrally depresses the respiratory rhythm generator of neonatal mice.</a> Neuroscience. 170, 1098-1109 (2010).</li><li>Arata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+activity+of+the+neonatal+dorsolateral+pons+in+vitro.">Respiratory activity of the neonatal dorsolateral pons in vitro.</a> Respir Physiol Neurobiol. 168, 144-152 (2009).</li><li>Onimaru, H., Homma, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+functional+neuron+group+for+respiratory+rhythm+generation+in+the+ventral+medulla.">A novel functional neuron group for respiratory rhythm generation in the ventral medulla.</a> J Neurosci. 23, 1478-1486 (2003).</li><li>St-John, W. M., Paton, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizations+of+eupnea,+apneusis+and+gasping+in+a+perfused+rat+preparation.">Characterizations of eupnea, apneusis and gasping in a perfused rat preparation.</a> Respir Physiol. 123, 201-213 (2000).</li></ol>

The authors have no competing financial interests or conflicts of interests to disclose.

Solunum aktivitesinin doğru miktar zor olabilir. Nitekim, nefes otomatik ve gönüllü hem de olabilecek bir fonksiyondur ve bu ortamda, vücudun ihtiyaçlarına duygusal durumu ve davranış göre modüle edilir. Bu tekniğin avantajı, solunum komutu üretmekten sorumlu nöral elemanların izolasyondur. Bu nedenle, beyin sapı-omurilik preparatları ve pletismografi elektrofizyolojik ölçümler sırasıyla in vitro ve in vivo olarak bütün nöronal solunum ağ çalışma tamamlayıcı tekniklerdir. Beyin s...

Solunum dioksijen (O 2) alımı ve karbondioksit (CO 2) eliminasyon izin beyin tarafından kontrol edilen karmaşık ve hayati faaliyeti vardır. Merkezi solunum sürücü hem memelilerde 1 beyin sapı bulunan karmaşık bir ağ, amfibiler 2, sürüngenler 3, kuşlar ve balıklar 4 5 tarafından oluşturulur. Nefes çalışma in vivo olarak işlenebilir bile, hassas mekanik araştırmalar solunum kontrolü ağına doğrudan erişim gerektirir. Bu amaçla, Adrian ve Buytendijk beyin yüzeyi kayıt solungaç havalandırma 5 ile bağlantılı üretilmiş bir ritm yerleştirilmiş elektrodlar olan bir düşük akvaryum balığı hazırlama, geliştirdi. Bu yaklaşım sonradan doğan kemirgenler kullanılmak üzere 1984 yılında 6 Suzue tarafından uyarlandı. Bu hazırlık gelişi solunum nörobiyoloji önemli ilerlemelere yol açmıştır. Bu, nispeten basit olduğundan, bu teknik h sunulmaktadırere ritmik motor davranışlar ve yenidoğan kemirgenlerde kökenleri temel araştırmaların geniş bir mükellef olduğunu. Bu yöntemin genel amacı inspiratuar aktivite nöral korelasyonu kaydetmek için, bir solunum benzeri ritim solunum ağı tarafından üretilen kurgusal nefes çağırdı. Bu yöntem, hem de vahşi tip 7 ve transjenik 8 hayvan solunum varyasyonlar ve farmakoloji inspiratuar yanıtları hedef araştırma amaçları geniş bir aralığında kullanılabilmektedir. Kaydedilen sinyalin fizyolojik alaka ilgili deneyler duyusal aferentlerde olmadan, düşük bir sıcaklıkta gerçekleştirilir ve aCSF içinde glikoz ve O 2 konsantrasyonları yüksek olduğu koşullarda, sorular gündeme getirilmiştir göz önüne alındığında. In vivo ve in vitro koşullarda arasında belirgin farklılıklar olmakla birlikte (örn., Inspiratuar patlamaların sıklığı) gerçeği varlığı kalırSolunum ağının 6 temel unsurları mümkün hayati homeostatik işlevi 9,10 ilişkili sağlam bir ritim eğitimi yapmak. Geliştirilmesi ve bu tekniğin kullanılması ardındaki mantık, özellikle yenidoğanlarda, in vivo pek erişilebilir solunum şebekenin beyin sapı elemanları, doğrudan erişim kolaylaştırmaktır. Beyin sapı sıkı kontrol koşulları altında yerleştirilir: kaydedilen ritim akciğerlerde veya karotid organları periferik afferent girdiler modüle değil, çalışma merkezi solunum sürücünün kendisini 11 odaklanmasını sağlar. Bu nedenle, bu erişim uyaranlara uygulanır ve çıkış sinyalini kaydetmek için kullanılır. Kayıtları, pletismografi aksine, solunum ritminde vücut boyunca tüm bileşenleri ile modüle (örn., Akciğer şişliği, periferal chemosensors), zor hassas uyaranlara uygulamak adrestir. Bir Inewborn sıçan, protokol yapay beyin-omurilik sıvısı (aCSF) muhafaza izole bir beyin sapında dördüncü ventral kök sinyali ve bir kesik omuriliği, kayıt oluşmaktadır. Beyin-omurilik preparasyonlar tarafından oluşturulan ritim inspiratuar sinyali 9 bağlanmış, tek tek ağır patlamaları oluşmaktadır. 7 - İzole beyin sapı-omurilik hazırlıkları kolayca 4 (P4 P0) post-natal 0. günden sıçanlarda kaydedilebilen vardır. Bu yaklaşım, genellikle, solunum ağın hipoksik yanıtı ve aynı zamanda hiperkapni, asidoz ve ilaçlara yanıtını değerlendirmek için kullanılır. Akut hipoksi protokol sunulmuştur. Bu uyarım aCSF içinde O 2 çekilmesi ile elde edilir; Bu yaklaşım genellikle hipoksiktir hoşgörü ve yanıt değerlendirmek için kullanılır. Protokol hipoksi maruziyeti sonunda (Şekil 1) 12 kadar ilk dakikadan itibaren bir ritim depresyon neden olur. Bu depresyon tersine çevrilirPost-hipoksik iyileşme 12&#39;ye sırasında. Deneysel tasarım ile ilgili, beyin sapı rostral kısmında bulunan pons, ritim jeneratörü 8 üzerinde inhibitör etkiye sahip olduğunu fark etmek önemlidir. Böylece, tam beyin sapı ve rostral omurilik hazırlıklar daha düşük bir ritim gösterir. Kayıt için izole numunede pons dahil edilmesi Deney 13 hedefine göre belirlenir; soğanilik ağdaki pons etkisi çalışma sonuçlarını 14 karşılaştırmak pons olan ve olmayan kayıtları gerektirecektir. Ayrıca, bu tekniğin avantajlarından biri, mezensefalik ve / veya diensefalik bölgeleri 15,16 dahil hazırlık rostral bir kısmının etrafında uzanan mümkün ponto-medüller solunum ağda bu bölgelerin etkisini değerlendirmek için yapma imkanı vardır.

<table><tbody><tr><td>Sylgard</td><td>Sigma Aldrich</td><td>761036-5EA</td><td>Use under hood</td></tr><tr><td>NaCl</td><td>Bioshop</td><td>SOD002</td><td></td></tr><tr><td>KCl</td><td>Bioshop</td><td>POC888</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>CCL444</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>MAG510</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Bioshop</td><td>SPM306</td><td></td></tr><tr><td>D-glucose</td><td>Bioshop</td><td>GLU501</td><td></td></tr><tr><td>Carbogen</td><td>Linde</td><td>343-02-0006&amp;nbsp;</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments, Hamden, CT, USA</td><td>TC-324B</td><td></td></tr><tr><td>Suction electrode</td><td>A-M Systems, Everett, WA, USA</td><td>model 573000</td><td></td></tr><tr><td>Differential AC amplifier</td><td>A-M Systems, Everett, WA, USA</td><td>model 1700</td><td></td></tr><tr><td>Moving averager</td><td>CWE, Ardmore, PA, USA</td><td>model MA-821</td><td></td></tr><tr><td>Data acquisition system</td><td>Dataq Instruments, Akron, OH, USA</td><td>model DI-720</td><td></td></tr><tr><td>LabChart software</td><td>ADInstruments, Colorado Springs, CO, USA</td><td></td><td></td></tr><tr><td>Prism sofware</td><td>Graphpad, La Jolla, CA, USA</td><td></td><td></td></tr><tr><td>Dissection chamber</td><td>Plastic box (e.g. petri box) will do</td><td></td><td></td></tr><tr><td>Recording chamber</td><td>Home made</td><td></td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Micromanipulator</td><td>World Precision Instrument Inc, Sarasota, FL, USA</td><td>KITE-R</td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Peristaltic pump</td><td>Gilson, Middleton, WI, USA</td><td>MINIPULS 3</td><td></td></tr><tr><td>Faraday Cage</td><td>Home made</td><td></td><td></td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr></tbody></table>

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.

Girişte belirtildiği gibi, bu tekniğin en önemli avantajlarından biri beyin sapı doğrudan erişim çeşitli uyaranlara uygulamaktır. Bir örnek olarak, hipoksi burada uygulandı. Şekil 1. AB hem normoksik ve hipoksik koşullarda, bir tam protokolü kayıt gösterir. Şekil 1.CE yani normoksik koşullar (kaydedilen ritmi görüntüler aCSF% 95 O2 ve% 5 ile kabarcıklandırılmış 26 ° C&#39;de CO2). Daha önce tam olarak bu hazırlama 11</su...

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

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

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 ...

electrophysiology-on-isolated-brainstem-spinal-cord-preparations-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.5K Görüntüleme.  Laval University. 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.

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

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 ...

Nörobilim

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Omurilik Elektrofizyoloji

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Biyoloji

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

Bir izoelektrik Beyin Devlet indüksiyon Nöronal Eksitabilite üzerinde Endojen Sinaptik Faaliyet etkisini araştırmak için<em&gt; İn Vivo</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

Yeni Bir Yöntemi Kullanarak Serbest Dolaşan Yenidoğan Sıçanlarında EEG Kaydı

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Akut<emIn vivo</em&gt; Anestezi Uygulanan Sıçanlarda Hiperdirect Pathway&#39;den Lokal Alan Potansiyellerinin Elektrofizyolojik Kayıtları ve Çoklu Ünite Aktivitesi

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Kortikal yerel alan potansiyelleri, Elektrokardiyogram, Electromyogram ve ritim serbestçe hareket bir sıçan gelen nefes eşzamanlı kayıt

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Ritmik aktif hazırlanması Vitro yenidoğan kemirgen beyin-omurilik ve ince dilim

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Farelerde beyin sapı uyarılmış yanıt odyometrisi veri edinme ve Analizi

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

Trans-Spinal Doğrudan Akım Stimülasyonu Sırasında Tip-Tanımlanmış Sıçan Spinal Motoneurons In Vivo Hücre İçi Kayıt

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

<meta charset="utf8"></head><body>Farelerde Derin Beyin Sapı Yapıları için Açılı Silikon Çoklu Elektrot Kayıtları

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

<meta charset="utf8"></head><body>Yazar Spotlight: Farelerde Anesteziye Bağlı Uyku Yollarının ve Nöronal Uyarılabilirliğin Araştırılması

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Kafası Sabit Farelerin Beyin Sapından Akut Tek Üniteli Çoklu Elektrot Kayıtları

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

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

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