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 border="0" cellpadding="0" cellspacing="0">
	<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>CaCl2</td>
			<td>Bioshop</td>
			<td>CCL444</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Bioshop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</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&#160;</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

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

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

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 ...

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

Multi-contact kullanarak Yerel Ağ İşleme incelenmesi Laminer Elektrot Kayıt

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

Kemirgenler davranmak yılında Hareketli Silikon Problar tarafından Nöronlar Büyük ölçekli Kaydı

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Kortikal Nöronlar In Vivo İki foton Of Görüntüleme Deneyimi bağımlı Moleküler Değişiklikler

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

Sinir Devre Kaydı bir Bozulmamış Hamamböceği Sinir Sistemi

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

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

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

Eşzamanlı Elektrofizyolojik Kayıt ve Suprakiazmatik Nucleus Nöronlar Kalsiyum Görüntüleme

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

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Mikro Elektrod Dizilerinde Nöronal Hücre Kültürlerinin Uyarılmasına Ağ Yanıtına Zamana Bağlı Artış

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...