We thank Dr. Larry D. Frye for editorial help with this manuscript. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 24300129 (to M.L.), 25890005 (to Y.O.) and 26640025 (to Y.T.), the National Agriculture and Food Research Organization (to Y.U.), the World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.O., Y.T., Y.U. and M.L.) and the Nestl&#233; Nutrition Council, Japan (to M.L.).

Etik Beyanı: Hayvan denekleri Prosedürler Tsukuba Üniversitesi Kurumsal Hayvan Deney Komitesi tarafından onaylanmıştır. EEG / EMG Recordings 1. Elektrotların hazırlanması ve Kablolar <ol><li> Aşağıdaki prosedüre göre EEG / EMG kayıt elektrot hazırlayın. Not: Elektrot tek ve 1 hayvan için sadece kullanılabilir. Dikkatlice tüm bağlantı için kablo yapılandırmasını planlayın. Doğru yönlendirme için konnektörleri işaretleri yerleştirin. <ol><li> 2 cm paslanmaz çelik tel bir 4 pinli başlığın her pimi lehimleyin. Kısaca, pin telin bir ucunu tutun tel-pin eklem üzerine sıcak havyaya yerleştirin ve bu sadece yeterli lehim eklem içine sorunsuz sağlamak için bazı lehim eriyerek. Pin çok fazla ısı uygulamak için dikkatli olun; Aksi takdirde, pimleri etrafında plastik eriyecek. </li><li> Kafasına iğne başlığına bağlı 2 teller her serbest ucunu lehim1.0 mm çapında paslanmaz çelik vida. Kısaca, vida başının altına Konuya telin serbest ucunu tutun tel vida eklem üzerine sıcak havyaya yerleştirin ve bu sadece yeterli lehim eklem içine sorunsuz sağlamak için bazı lehim eriyerek. Vida olmadan 2 teller EMG kayıt elektrotları olarak hizmet ise vidalarla 2 tel, EEG kayıt elektrotları olarak hizmet vermektedir. </li><li> EMG sinyalinin kalitesini artırmak için EMG elektrotları sonunda yalıtım 1 mm kapalı şerit makas kullanın. </li><li> Tamamen EEG / EMG kayıtları sırasında elektrik gürültüsünü azaltmak için ince bir tahta sopa ya da diş çekme kullanılarak epoksi yapıştırıcı ile tüm lehimli işaretçilerine kapsamaktadır. </li></ol></li><li> Aşağıda tarif edildiği gibi kayma halkasına elektrodu bağlamak için bir kablo hazırlayın. Bu kablo, yeniden kullanılabilir. <ol><li> 30-cm düz kablonun bir tel ile bir 4-pin FFC / FPC bağlacının her iki pimi lehimleyin. Kısaca, pin tel soyulmuş ucunu tutun sıcak havyaya yerleştirmekTel-pin eklem üzerine ve yeterli lehim eklem içine sorunsuz bir şekilde yürütülebilmesi için bazı lehim eriyerek. Not: EEG / EMG kayıtları için kullanılan deneysel hayvan kafes yüksekliği için uygun olan yassı kablo uzunluğunu seçin. </li><li> Lehim düz kablonun diğer ucundaki bir tel ucuna yuva kıvrım. Kısaca, tel elimden serbest ucuna bir kıvrım soket tutun tel soket eklem üzerine sıcak havyaya yerleştirin ve bu sadece yeterli lehim eklem içine sorunsuz sağlamak için bazı lehim eriyerek. </li><li> Her bir mahfaza kıvrım 4-konumuna yuva kıvrım yerleştirin. </li><li> Tamamen ince tahta sopa ya da diş çekme kullanılarak epoksi yapıştırıcı ile yuva kıvrım kapsamaktadır. </li></ol></li></ol> Fare Head Elektrotların 2. implantasyonu (Süre:. Yaklaşık 20 dk) <ol><li> Ameliyat öncesi sıcak bir boncuk sterilizatör tüm cerrahi aletler sterilize edin. Bir erkek fare anestezisi (10-20 haftalık, 20-30 g)pentobarbital intraperitonal enjeksiyonu (50 mg / kg) ile. Fare derinden bir ayak kısma anestezi olduğunu kontrol ettikten sonra, kesme makineleri ile baş ve boyun üzerinde saç tıraş. </li><li> Stereotaksik çerçeve için fareyi hareket ettirin ve 2 kulak çubukları arasındaki kafa düzeltmek. Kuruluğunu önlemek için gözleri vazelin sürün. Alkol ile traş-cildi temizlemek ve kafatası açığa çıkarmak için bir neşter ile orta hat boyunca kesmek. Açık cerrahi alana tutmak için cildi Clip. </li><li> (Matkap 2 delik kafatası içine, frontal kortikal alana bir (bregmaya 1 mm anterior, orta hatta 1,5 mm lateral) ve parietal alan üzerinde diğer: karbür kesici (0.8-mm çaplı matkap boyutu) kullanarak Paxinos ve Franklin stereotaksik koordinatlarına göre sağ yarımkürenin lambda 1 mm anterior lateral 1,5 mm orta hat için). 23 </li><li> Her bağırıyorum 2.5 döner - bir kuyumcuya vida sürücüsünü kullanarak, delikleri yer paslanmaz çelik EEG kayıt vida ve 2 yapmakkorteks üzerinde epidural konumlandırma için w. Not: beyin dokusunun zarar görmesini önlemek için çok derin vidayı takmayın. Vidalar sıkıca kafatası sabit olup olmadığını kontrol edin. Bu çoklu kayıtları (genellikle en fazla 1 ay) uzun bir süre boyunca sabit EEG sinyallerini olması önemlidir. Wiggly vidaları EEG eserler üretmek ve deneysel programının sona ermesinden önce kapalı gelebilir. </li><li> Elektrot düzeneğini Fix kafatası anında yapıştırıcı ile (bkz Bölüm 1.1, pim yukarı döndü) ve diş çimento ile kaplayın. Trapez (boyun) kaslar forseps ile bilateral küçük delik açın ve deliklere EMG elektrotları olarak hizmet paslanmaz çelik teller yerleştirin. Kas maruz kalmasını önlemek için ipek iplik (0.1 mm çap) ile cilt dikin. </li><li> Stereotaksik çerçeve fareyi çıkarın. Bakteriyel enfeksiyonu önlemek için fare, ampisilin (100 mg / kg) ve intraperitoneal olarak meloksikam (1 mg / kg) yönetir ve sırasıyla ameliyat sonrası ağrıyı azaltmak için. Keısı yastığı üzerinde fare ep ve sternum yatma korumak için yeterli bilinci yerine kadar onu izleriz. Ev ayrı kurtarma sırasında fare diğer hayvanlar tarafından elektrotların kaldırılmasını önlemek ve ameliyat sonrası birinci günde intraperitoneal meloksikam (1 mg / kg) yönetmek için. </li></ol> 3. Kayıt ve kavranması EEG / EMG Veri <ol><li> 1 haftalık iyileşme dönemi, ev ses geçirmez bir kayıt odasına yerleştirilen deneysel kafeste tek tek her bir fare sonra. 12 saat ışık / 12 saat karanlık döngüleri kontrol otomatik olarak 23 ± 1 ° C ortam sıcaklığı korumak ve (aydınlatma yoğunluğu ~ 100 lux, 08:00 ışıklar). </li><li> Bir kayıt kablosu fare kafasına EEG / EMG elektrot düzeneğini takın. Kayıt kablosu (fare hareketleri kablo büküm ile sınırlı değildir böylece tasarlanmış) bir kayma halkası ve EEG / EMG sinyal yükseltici bağlı olduğundan emin olun. Filtre EEG / EMG sinyalleri (EEG, 0,5-64 Hz, EMG, 16-64 Hz), bir analog-dijital dönüştürücü (A / D) 128 Hz örnekleme hızında dijital ortama aktarmak ve nihayet EEG / EMG kayıt yazılımını çalıştıran bir bilgisayarda (Table &#39;Malzeme&#39; 4. girişi üzerine kayıt alttan). </li><li> Kayıt odasına 3 gün - 2 için fare alıştırmak. EEG / EMG kayıt intraperitoneal ilaç yönetimleri içeriyorsa, yavaşça ilaç uygulama sırasında her alışkanlık gününde fareyi anlaştım. </li><li> Daha sonra, EEG / EMG kayıt yazılımı (Table &#39;Materyalleri&#39;, alttan 4. entry) başlatın. <ol><li> &#39;Veri dosya bilgileri&#39; sekmesini seçin ve dosya adının yanındaki kutuyu tıklatın. Bir dosya adı girin ve &#39;Kaydet&#39;i tıklayın. </li><li> &#39;Kayıt koşul&#39; sekmesini seçin ve kaydedilecek tüm EEG / EMG kanalları seçmek. </li><li> &#39;Kayıt koşulu&#39; sekmesinde örnekleme frekansı (128 Hz) seçin. </li><li> Seçilen kanallar inci düzgün görüntülenen olup olmadığını kontrol edine &#39;Kanal bilgisi&#39; sekmesi. </li><li> &#39;Timer ayarı&#39; sekmesini seçin ve EEG ve EMG görüntülemek için &#39;Monitor&#39; tıklayın. </li><li> EEG / EMG sinyalleri doğru görüntülenir olup olmadığını kontrol edin. </li><li> &#39;Timer ayarı&#39; sekmesini seçin ve &#39;Ana Timer&#39; alanında kaydın başlangıcı ve sonu için saat saatini ayarlayın. </li><li> Kayıt işlemini başlatmak için &#39;Zamanlayıcı ayarı&#39; sekmesinde &#39;Monitor&#39; tıklayın. </li></ol></li><li> Başlangıçta altında Tutanak EEG / EMG sinyalleri (yani., Serbestçe davranmak fare uyku / uyanıklık davranışı) ve farklı tedavi koşulları (örn., Kafein idaresi veya sahte tedavisi) birkaç gün içinde. Son deney gününde sonra, pentobarbital intraperitonal enjeksiyon ile (200 mg / kg) ile fare öldürülür. </li></ol> EEG / EMG Veri dayanarak Davranış Devletin 4. Puanlama <ol><li> EEG / EMG analizi için yazılımını başlatın (Table &#39;Malzeme&#39;, alttan 4. girişi). 3. adımda altında üretilen EEG / EMG ham verileri (.kcd dosyası) açın &#39;Sleep&#39;in&#39; sekmesini tıklayın ve Epoch zamanı seçin. 10 sn seçin. </li><li> &#39;Sleep&#39;in&#39; sekmesini tıklayın ve otomatik olarak 3 aşamaya 10 sn dönemini puanı &#39;Multi-tarama&#39; seçeneğini (yani., NREM ve REM uyku, uyanıklık ve) EEG ve EMG ve güç spektral analizi genliklerinin temelinde EEG. 3 </li><li> &#39;EEG için FFT koşulu tıklayın. </li><li> Güç spektral analiz parametrelerini ayarlayın [256 datum noktaları, Hanning pencere fonksiyonu ve çağın başına 5 spektrumları ortalamasına (2 EEG sn karşılık gelen)]. 3 &#39;Tamam&#39;ı tıklayın. </li><li> Tıklayın, otomatik tarama başlatmak için &#39;Tarama Başlat&#39;. Attı verileri (.raf dosyası) açın. </li><li> Temsilcisi Sonuçlar Şekil 1B ve Tablo 1 (otomatik tarama sonuçlarını kontrol edin ve gerekirse standart kriterlere dayalı elle düzeltin </strong&gt;). 2, 3 Kısaca, tıklayın ve bir yanlış sonucu çağında sol fare tuşunu basılı tutun ve yanlış sonucu dönemini dize boyunca imleci sürükleyin. Sol fare tuşunu bırakın ve pop-up pencerede doğru davranış durumu seçin. Not: Bazen, iki uyanık devletler arasındaki geçişte dönemini, bir devlete açıkça puanı zordur. Bu gibi durumlarda, dönem görünüşte duruma atılır olmalı ve aynı kriterler verileri yeniden üretilebilirlik sağlamak için deney boyunca benzer dönemini uygulanmalıdır. </li></ol>

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Y., 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=Piromelatine+exerts+antinociceptive+effect+via+melatonin,+opioid,+and+5HT1A+receptors+and+hypnotic+effect+via+melatonin+receptors+in+a+mouse+model+of+neuropathic+pain.">Piromelatine exerts antinociceptive effect via melatonin, opioid, and 5HT1A receptors and hypnotic effect via melatonin receptors in a mouse model of neuropathic pain.</a> Psychopharmacol. 231 (20), 3973-3985 (2014).</li><li>Qu, W. M., 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=Lipocalin-type+prostaglandin+D+synthase+produces+prostaglandin+D2+involved+in+regulation+of+physiological+sleep.">Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep.</a> Proc. Natl. Acad. Sci. USA. 103 (47), 17949-17954 (2006).</li></ol>

The authors Yujiro Tauguchi and Sayaka Kohtoh are employees of Kissei Comtech Co., Ltd that develops SleepSign software for acquisition and automatic scoring of EEG/EMG data used in this article.

Bu protokol, düşük gürültü, düşük maliyetli ve yüksek verimli koşullarda uyku ve uyanıklık değerlendirilmesini sağlar EEG / EMG kayıtları için bir set-up açıklar. Nedeniyle EEG / EMG elektrot kafa grubunun küçük boyutu, bu sistem fare içine ilaçların optogenetics (fiber optik implantasyon) ya da, birlikte eş zamanlı kanül implantasyonu ile, mikroinfüzyon dahil içi beyin deneyleri için diğer implantlar ile kombine edilebilir İlave elektrik sinyalleri (örn., kontralateral EEG, e...

Teknik gelişmeler sık ​​sık nörobiyolojik süreçler anlayış atılımlar çöktürülmüş var. Örneğin, insan derisi kaydedilen elektrik potansiyelleri sinüs dalgalarının şeklini aldı 1929 yılında Hans Berger keşif, sıklığı, doğrudan konunun uyanıklık düzeyi ile ilgilidir, uyku-uyanıklık anlayışı hızlı ilerlemelere yol açmıştır hem hayvanlarda ve insanlarda hem de düzenleme. 1 Bu gün electroencephlogram (EEG) için, elektromyogram (EMG), yani. ile birlikte, iskelet kasları tarafından üretilen elektriksel aktivite, neredeyse her deneysel ve klinik verileri &quot;omurga&quot; temsil insanlar dahil, hayvanlar davranıyor kortikal nöronların aktivitesi ile davranış ve fizyoloji ilişkilendirmek istiyor değerlendirmesi. En temel uyku araştırma laboratuvarlarında bu EEG kayıtları d edinilen burada bir kablo tabanlı sistemi (Şekil 1) kullanılarak gerçekleştirilenata desen ve spektrum analizi off-line tabi [örneğin., bir hızlı Fourier (FFT) algoritması dönüşümü uygulayarak] konunun uyanıklık durumu kaydediliyor belirlemek için. 2, 3 Uyku hızlı göz hareketi oluşur (REM) ve REM olmayan (NREM) uyku. REM uykusu hızlı bir düşük voltaj EEG, rastgele göz hareketi ve kas atonia, kaslar etkin bir felç olduğu bir devlet ile karakterizedir. Vücut beyinden ölçüde kesilir ve derin uykuda gibi görünüyor, oysa beyin aktivitesi, bu uyanıklık benzer, çünkü REM uykusunda da, paradoksal uyku olarak bilinir. Buna karşılık, motor nöronlar NREM uyku sırasında uyarılır ama hiçbir göz hareketi yoktur. EEG 4 Hz - İnsan NREM uykusu evre 4 derin uyku veya yavaş dalga uykusu denir ve 0.5 arasındaki delta aktivitesi ile Büyük, yavaş beyin dalgaları ile tanımlanır sayede 4 aşamada, ayrılabilir. Öte yandan, sıçanlar a gibi küçük hayvanlarda NREM uykusu fazlar arasında, bir alt bölümü,nd fareler, insanlarda görüldüğü gibi onlar uyku uzun konsolide dönemleri yok çünkü çoğunlukla kurulmamıştır. Yıllar geçtikçe, ve EEG yorumuna dayanarak, uyku-uyanıklık düzenlemenin çeşitli modeller, devre ve humoral tabanlı hem de ileri sürülmüştür. Nöral ve uyku ya da alternatif olarak, gerek hücresel temeli &quot;uyku sürücü&quot; çözümsüz kalır, ama uyanık döneminde inşa eder ve uyku ile harcanmış bir homeostatik basıncı olarak kavramsallaştırma edilmiştir. Bir teoriye göre endojen somnogenic faktörler uyanıklık sırasında ve kademeli birikimi uyku homeostatik basınç destekleyen olduğunu birikir olmasıdır. Humoral faktörler tarafından düzenlenir uyku ilk resmi hipotez 1892 4 yayınlanan Rosenbaum&#39;un çalışmaları alacak olsa da, bu Ishimori 5, 6 ve bağımsız pieron 7 oldu ve 100 yıl önce, uyku teşvik kimyasalların varlığını gösterdi. Her iki araştırmacılar hypnogenic maddeler veya &#39;hypnotoxins&#39; uykusuz köpeklerin beyin omurilik sıvısı (BOS) mevcut olduğunu, önerilen ve gerçekten kanıtladı. 8 Geçtiğimiz yüzyılda uyku homeostatik süreçte rol birkaç ek varsayımsal hypnogenic maddeler tespit edilmiştir (inceleme için, ref. 9), prostaglandin (PG) D 2, 10, sitokinler, adenosin 11, 12 anandamid, 13 ve Ürotensin II peptidi de dahil olmak üzere. 14 Belli bir dereceye kadar uyku ve uyanıklık ve devre tabanlı teoriler ilham erken ve orta-20 yüzyılda üretilen bulgular Economo 15, 16, MORUZZI ve Magoun 17, ve diğerleri tarafından deneysel çalışmalar, daha sonra hakim hümoral teorisini gölgesinde uyku. Bugüne kadar, birkaç &quot;Devre modeller&quot; Her (inceleme için, ref. 18) nitelik ve nicelik değişen verilerle haberdar öne sürülmüştür. Bir modelÖrneğin, yavaş dalga uykusu bazal ön, bir alan esas olarak Broca diyagonal bant ve substantia inominata yatay bacağının çekirdeğinin consisiting kolinerjik nöronların asetilkolin salımının adenosin aracılı inhibisyonu yoluyla oluşturulur önermektedir. uyku / uyanıklık regülasyonu 19 başka popüler bir model hipotalamus ve beyin sapındaki ventrolateral preoptik alanda uyku uyaran nöronlar arasındaki karşılıklı etkileşim ve inhibitör uyandırma uyaran nöronlar temelinde bir flip-flop anahtar mekanizmasını anlatır. 18, 20, 21 Ayrıca, ve REM uykusu üzerinden geçiş için, benzer bir karşılıklı etkileşim önleyici beyin sapında alanlar için önerilmiştir, bu ventral periakuaduktal gri yanal tegmentumda ve sublaterodorsal çekirdektir. 22 Toplu olarak, bu modeller değerli olduğu kanıtlanmıştır heuristics ve uyku araştırma çalışmaları için tanınan önemli açıklayıcı çerçeveler; Bununla birlikte, bir yeuyku-uyanıklık döngüsünü düzenleyen moleküler mekanizmaları ve devrelerin t dolgun anlayışı bileşenlerinin daha eksiksiz bir bilgi gerektirir. Aşağıda ayrıntılı Basım kayıt için sistem bu hedefe yardımcı olmalıdır.

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">4-pin header</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">A3B-4PA-2DSA(71)</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Ampicillin</td>
			<td style="width:143px;">Meiji Seika</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analog-to-digital converter</td>
			<td style="width:143px;">Contec</td>
			<td style="width:167px;">AD16-16U(PCIEV)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Caffeine</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">C0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbide cutter</td>
			<td style="width:143px;">Minitor</td>
			<td style="width:167px;">B1055</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Crimp housing</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-4DS-2C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Crimp socket</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-30SC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dental cement (Toughron Rebase)</td>
			<td style="width:143px;">Miki Chemical Product</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epoxy adhesive</td>
			<td style="width:143px;">Konishi</td>
			<td style="width:167px;">#16351</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">FFC/FPC connector</td>
			<td style="width:143px;">Honda Tsushin Kogyo</td>
			<td style="width:167px;">FFC-10BMEP1(B)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flat cable</td>
			<td style="width:143px;">Hitachi Cable</td>
			<td style="width:167px;">20528-ST LF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Instant glue (Aron Alpha A)</td>
			<td style="width:143px;">Toagosei</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Meloxicam</td>
			<td style="width:143px;">Boehringer Ingelheim</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital</td>
			<td style="width:143px;">Kyoritsu Seiyaku</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal amplifier</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sleep recording chamber</td>
			<td style="width:143px;">APL</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SleepSign software</td>
			<td style="width:143px;">Kissei Comtec</td>
			<td style="width:167px;">N/A</td>
			<td>for EEG/EMG recording/analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slip ring</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel screw</td>
			<td style="width:143px;">Yamazaki</td>
			<td style="width:167px;">N/A</td>
			<td>&#966;1.0&#215;2.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel wire</td>
			<td style="width:143px;">Cooner Wire</td>
			<td style="width:167px;">AS633</td>
			<td></td>
		</tr>
	</tbody>
</table>

polygraphic recording procedure for measuring sleep in mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

Şekil 1B farklı uyanıklık durumlarında fare EEG örneklerini göstermektedir. Tablo 1&#39;de görüldüğü gibi EEG 4 Hz altındaki bir delta ritimle büyük, yavaş beyin dalgaları gösterir ve EMG sadece zayıf ya da hiç sinyal varsa, dönemini NREM uyku olarak sınıflandırılır. EEG 6 ve 10 Hz arasındaki teta aralığında hızlı alçak gerilim beyin dalgaları gösterir ve EMG düşük genlik gösterirse dönemini REM uykusu olarak sın?...

Watch this Scientific Journal Video about Polygraphic Recording Procedure for Measuring Sleep in Mice at JoVE.com

Polygraphic Recording Procedure for Measuring Sleep in Mice

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Farelerde Uyku Ölçüm Basım Kayıt Prosedürü

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

polygraphic-recording-procedure-for-measuring-sleep-in-mice

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23.3K Görüntüleme.  University of Tsukuba. The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Video: Polygraphic Recording Procedure for Measuring Sleep in Mice

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Farelerde Motor Koordinasyon Ölçüm

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Nörobilim

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 ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Kemirgenler Faaliyet teker Running kayıt ve Sirkadiyen Rhythms Analizi

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Dorsal Kök Potansiyel Kayıt By Farelerde Spinal Presinaptik Engellenme Ölçme<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Fareler Soğuk Hassasiyeti ve Uyum belirlenmesi için Basit ve ucuz Yöntem

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Uyanık Farelerde Nöroaktif Maddelerin Microiontophoretic Uygulaması ile birlikte Nöronal Faaliyet dışı Kaydı

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Ürünleri ve tüm hücre yama kelepçe kayıt Xenopus laevis Tectal nöronlar için iletişim kuralları

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

Dağınık şekilde kayıt sınırlı salınım oluşur içinde fareler davranmak

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

Farelerde uyku/uyanıklık durumları Izleme sırasında nöral devrelerin optogenetik manipülasyon

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

Fareler Ikincil motor korteks bilateral lokal saha potansiyel kayıt ile Yarımküre lateralizasyon değerlendirilmesi

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

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