Bu eser Doğa Bilimleri Pekin Vakfı (No.7162124) ve Beijing Üniversitesi Çin tıbbı Xin-ao Vakfı tarafından desteklenmiştir.

Bütün hayvanlar kılavuzlarınıza uygun olarak etik kullanım ve Laboratuvar hayvanlarının bakımı Çin mevzuat tarafından muhafaza.
1. ön modelleme hazırlık
<ol><li>
Laboratuvar hazırlık
	<ol>
<li>UV lambası en az 30 dk önce deneme için çalıştırın.</li>
		<li>25 ± 3 ° C ve bağıl nem oranı yaklaşık % 30 laboratuvar sıcaklık kontrol.</li>
		<li>6: 00'de ışık laboratuvar açmak ve 18:00 12 h/12 h açık/koyu döngüsü kurmak için devre dışı.</li>
	</ol>
</li>
	<li>
Birden çok Platform kutusu İnşaat
	<ol>
<li>Bir opak plastik tüpü 110 × 60 × 40 cm3kapak olmadan oluşturun.</li>
		<li>On beş dairesel platformlar düzeltmek (h = 8 cm, d = 6.5 cm) tank dibinde, hangi düzenli dağıtmak üç satır ve 5 sütun. Yeterli alan her platform arasında kabaca 10 cm arasında sütunlar ve satırlar arasında 13 cm bırakın.</li>
		<li>Suyu çıkışı tank yan tarafında ayarlayın ve bir musluk yüklemek.</li>
		<li>Bir demir-tel örgü kapak tankları için üzerinde asılı bir yemek kutusu ile yapmak.</li>
	</ol>
</li>
	<li>
Gruplandırma ve konut fareler Not: Wistar erkek rats 8 hafta yaşlı, yaklaşık 200-210 g ağırlığında denemeye kullanılır. Fareler model oluşturma işlemi sırasında gruplar içinde yaşar.<ol>
<li>Rats kuyruk kökleri bir marker kalem ile numara.</li>
		<li>Fareler tartmak, son derece hafif veya ağır olanları hariç tutmak ve diğer rasgele model ve denetimi gruba ayırın.</li>
		<li>Fareler hafifçe temiz kafes koyun ve en az 3 gün için laboratuara alışmana izin. Yeterli su ve besin kaynağı sağlar.</li>
	</ol>
</li>
</ol>2. MMPM ile modelleme
Not: Süreci 18: 00'de başlıyor ve 21 gün boyunca günde 14 saat olmak üzere toplam ertesi gün saat 8:00 sona erer. Girişim faktörlerini önlemek için aynı kişi aynı önlük giyen süre tüm deney yapmak için gereklidir. 10 Wistar rat denemede kullanılır.
<ol><li>Tank yer düz bir yüzey, Örneğin, zemin üzerinde. Daha sonra tank yaklaşık 7 cm sıcak (25 ± 3 ° C) su, yaklaşık 1 cm düz platformu ile doldurun.</li>
	<li>İçinde belgili tanımlık tank tüm rats için yeterli yiyecek ve içecek 1 gün için hazır olun. Yem ve su gıda kutusu koymak ve kapağında asın. Not: Bazı akıllı fareler gıda kutusu üzerinde dinlenmek bilgi edinin. Eğer öyleyse, onları tanka geri götürmek.</li>
	<li>Model grubu fareler dışarı kafesin, onu kuyruğundan kapmak alıp hafifçe tank içine koy. Su su korkusu motive etmek için platformlar yerine tüm rats başlatın. Her fare fareler denetim grubunun yeterli yiyecek ve su ile orijinal onların kafes içinde kalırken, durmak için bir platform aldığından emin ol.</li>
	<li>Tank kapağı. Kaza önlemek farelerin izlemek. Bir sıçan daha 1 h için suda platform üzerine tırmanma olmadan kalırsa, havuzdan al ve testten kaldırmak.</li>
	<li>14 h sonra modeli fareler havuzdan al ve saçlarını ile bir kurutma makinası. Bu kaybolur, rats kuyrukları yeniden işaretleyin. Fareler için özgün onların kafes dönmek ve yeterli yiyecek ve su ile sağlamak.</li>
	<li>Tank her köşesine saklandığı yerden. Tank bir tarafı yüceltmek ve musluk kanalizasyon outflow için açın.</li>
	<li>% 75 etanol sprey tüpüyle sterilize ve UV ışığına maruz kalmaktadır.</li>
</ol>3. model değerlendirme: Davranış testi
Not: Tüm testler davranış laboratuarda uygulanmaktadır. Gürültü ve fazladan ışık test sırasında rahatsızlık önlemek için izin verilmez. Mümkünse, aynı kişinin kartına programın kopyalamasını her testini gerçekleştirmek için kullanın. Karanlık bir ceket ve eldiven gri ölçek tanıma görüntü işleme için gereklidir. ÇOK gerçekleştirmek ilk fare davranışı üzerinde en az etkiye sahiptir.
<ol><li>
SIK SIK
	<ol>
<li>Kaydedici düzgün iş istasyonuna bağlı olan ve kutusunun her köşesinde kapsar emin olmak için açık alan kutuyu kontrol edin. Aydınlatma gölgeler kutusunda ortadan kaldırmak için ayarlama yapar.</li>
		<li>Özgün onların kafes davranış Laboratuvarda fareler taşıyın. En az 1 saat önce test için alışmana izin.</li>
		<li>Temiz ve dışkı veya önceki deneyden yaptı koku yok sağlamak için % 75 etanol kutusuyla sterilize.</li>
		<li>Bir sıçan kafes sırtını tarafından çıkarın ve hafifçe kutusunun merkezi alanı koydu. Hızlı bir şekilde silah atış engellemek değil için kutusundan geri çekilin.</li>
		<li>Sıçan numarasını girin ve kayıt işlemini başlatmak. Sayısı ve kaydı fare dikey faaliyetleri, yetiştirme ve tırmanma gibi sıklığı.</li>
		<li>5 dk sonra kaydı durdurmak, fareyi kutudan almak ve kafese geri.</li>
		<li>Adımları 3.1.3 - tüm rats test bitinceye kadar 3.1.6 yineleyin.</li>
	</ol>
</li>
	<li>
EPM
	<ol>
<li>Çok (adım 3.1.1 - 3.1.2) gelince ön onay ve calıştıkları adımları gerçekleştirin.</li>
		<li>Bir sıçan kafes sırtını tarafından çıkarın ve yavaşça iki kolu kavşak parçası koydu. Fareyi sol açık kol doğru arazi ve hızlı bir şekilde terk atış engellemek değil için.</li>
		<li>Sıçan numarasını girin ve kayıt işlemini başlatmak. Sayısı ve kaydı farklı kol girişleri sıklığı. Fareyi test labirentinde kapalı düşerse alırım ve labirentine geri gönderin. Veri çözümlemeleri için ayrıntılı bilgileri kaydetmek.</li>
		<li>5 dk sonra kaydı durdurmak, fareyi çıkartıp almak ve kafese geri.</li>
		<li>Lanet çıkarın ve labirent eski sıçan kokusu ortadan kaldırmak için % 75 etanol ile silin.</li>
		<li>3.2.2 - tüm rats test bitinceye kadar 3.2.5 yineleyin.</li>
	</ol>
</li>
	<li>
ES testi
	<ol>
<li>Yüzme tankı (70 × 30 × 110 cm3) ile 80 cm (25 ± 3 ° C) sıcak su doldurun. Not: içinde belgili tanımlık tank bir termostat ise, su sıcaklığı yaklaşık 37 ° sıçan vücut ısısı için benzer C, ayarlanması gerekir. Eğer değilse, oda sıcaklığında sabit tutmak için ayarla.</li>
		<li>Her fare iğne ile demet bir yükleme yapmak ve yavaşça kendi kuyruk kök kravat. Yükü sıçan ağırlığı yüzde 10'u ağırlığındadır.</li>
		<li>Bir sıçan kuyruk tarafından Kepçe ve yüzme tankı atın. Fareler araya toplamak veya duvara sarılmak, onları ayrı ayarlamak ve onları suya geri götürmek.</li>
		<li>Zamanlama başlamak ne zaman fare koymak içine su ve bu yetersiz kaldığında dur zamanlama Şu anda hangi gösterdi dışarı su ağzı ve burnu daha--dan 10 için su altında ile mücadele için başarısızlık olarak s. Not: Bazen, bitkinlik ve boğulma aniden ortaya çıkar. Kaydetmek ve aynı zamanda hayvan kurtarmak için yeterli Denemecileri var emin olun.</li>
		<li>Sudan yorgun sıçanlar başkalarının kesintiye uğratmadan kaldırın. Saçlarını kuru, sayıları yeniden işaretleyin ve kafese geri gönderebilirsiniz.</li>
		<li>Bir grup tamamlandıktan sonra değişmek belgili tanımlık su içinde belgili tanımlık tank. Sonra tüm rats yapılır, yüzme tankı, boş ve temiz ve etanol ve UV ışığı ile sterilize.</li>
	</ol>
</li>
</ol>4. model değerlendirme: Merkezi sinir algılama
<ol><li>Bilinçsiz kadar % 10 kloral hidrat (3 mL/kg) intra periton enjeksiyon fareyle anestezi.</li>
	<li>Fareyi başını kesmek.</li>
	<li>Sonrası orta çizgi boyunca uzunlamasına bir kesi yapmak, iki tarafa da kafatası açın ve beyin ortaya çıkarmak. Kafatası çevirin, beyin kaldırmak ve beyin bir buz torbası koyun.</li>
	<li>Ayrı ve elmas şeklindeki alandır çevreleyen dokular ile açık bir sınır beyin tabanının orta kısmında hipotalamus kaldırın. Steril bir tüp içinde yerleştirin ve sıvı azot ile dondur. Tüm örnekleri-80 ° C buzdolabında saklayın.</li>
	<li>İçeriği 5-HT ve DA yüksek performanslı sıvı kromatografi (HPLC)8kullanarak hipotalamus algılamak.</li>
</ol>

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Yazarlar ifşa gerek yok.

MMPM aslında uyku yoksunluğu9için tasarlanmıştır. Fareler bir su tankına altta sabit platformları ile piyasaya sürüldü. Su içgüdüsel korkusu ile tahrik, fareler platformlarda ayakta kalır ve uyku yok oluşur. Çalışma farklı saatlik uyku yoksunluğu sıçan davranış ve ruh, tanıma bozukluğu10, negatif duygular11ve merkezi yorgunluk da dahil olmak üzere çeşitli değişikliklere yol gösterir. Bazı araştırmacılar kanıtlama...

Yorgunluk insan sağlığı1tehdit ana faktörlerden biridir. Son on yıl içinde yorgunluk periferik tetikleyen ama merkezi olarak tahrik ve her zaman eşlik etti duygusal ve bilişsel bozukluklar ile çeşitli araştırmalar kanıtlamıştır. İtalyan fizyolog A. Mosso ilk kelime merkezi yorgunluk2evlenme teklif etti. Genellikle sınırlı gönüllü faaliyet ve fonksiyon bozukluğu nedeniyle biliş bozulma impuls iletim merkezi sinir sistemi (MSS)3olarak tanımlanır. Periferik kas yorgunluğu ile karşılaştırıldığında, merkezi yorgunluk MSS, hem de bunun sonucunda duygusal/davranış bozuklukları, depresyon, anksiyete, biliş Engelli ve hafıza kaybı da dahil olmak üzere değişiklikleri vurgular. Bir çalışmada birçok faktör merkezi yorgunluk, hangi aşırı fiziksel aktivite ve zihinsel stres arasında oldukça vazgeçilmez4olduğunu tetikleyebilir gösterir. Patogenez gelince, triptofan-kynurenine yolu hipotez5 gibi teoriler bazı yollar değişiklikleri açıklamaya; Ancak, daha fazla ayrıntılı çalışmalar yine orta-periferik korelasyon merkezi yorgunluk ortaya çıkarmak gerekir.
Merkezi yorgunluk altta yatan mekanizma hala belirsiz olduğu için etkili bir hayvan modeli daha fazla araştırma için oldukça önemlidir. Varolan yorgunluk modelleri çoğunlukla koşu bandı6 ve yüzme ağırlık yüklü7, ruhsal etkenlere küçük endişe ile gibi aşırı egzersiz tarafından indüklenir. Daha iyi geliştirme merkezi yorgunluk benzetimini yapmak için bizim grup MMPM bir fare modeli geliştirdi. Model oluşturma işlemi sırasında fareler için uzun saat uyku süresi bölümü de dahil olmak üzere birden çok Platform kutusunda dar platformlarda ayakta kalır. Aşırı egzersiz modelleri farklı, MMPM modeli dikkate alınarak Merkez yorgunluk karmaşık patogenezinde zihinsel bir faktör olarak kısmi uyku yoksunluğu kullanır.
Modeli değerlendirme için biz çok ve EPM testleri anksiyete ruh hali ve gönüllü faaliyet belirlemek için kullanın. ES test periferik kas performansı ölçmek için yapılır. Buna ek olarak, biz sıçan beyin almak ve dopamin (DA) tespit / serotonin (5-HT) içeriği merkezi nörotransmitter farklılıkları gözlemlemek için her iki hypothalamuses.
Aşağıda sunulan iletişim kuralı modeli merkezi yorgunluk tekrarlanan fiziksel aktivite ve uyku, insan yaşamında yaygın bir durum taklit eksikliği tarafından indüklenen için tasarlanmıştır. Ancak, modeli süresini ayarlayarak, diğer birçok alanda gibi uyku gözlem ve stres çalışmalarda kullanılabilmesi için. Gelecekte araştırma, bu model daha fazla CNS değişiklikler ve onların bağlantı merkezi yorgunluk Patogenez mekanizması ortaya çıkarmak için periferik sistemiyle keşfetmek yardımcı olacağını umuyoruz.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>multiple platform sleep deprivation water tank</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>110cm x 60cm x 40cm. There are 15 plastic small platforms at the bottom. The small platform is 6.5cm in diameter and 8cm high</td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Beijing Weitong Lihua Experimental Animal Technology Company</td>
			<td>license number SYXK (Beijing) 2016-0011</td>
			<td>Use 32 Wistar healthy male rats ,8 week old (200-210 g)</td>
		</tr>
		<tr>
			<td>Agilent 1100LC high performance liquid chromatograph&#160;</td>
			<td>Agilent</td>
			<td>&#160;G1379A, G1311A, G1313A , G1316A&#160;</td>
			<td>&#160;G1379A, G1311A type chromatographic pump, G1313A automatic sampler, G1316A column temperature box</td>
		</tr>
		<tr>
			<td>DECADE II SDC electrochemical detector</td>
			<td>Dutch ANTEC company</td>
			<td></td>
			<td>glassy carbon electrode, Ag/AgCl reference electrode, workstations (Clarity CHS)</td>
		</tr>
		<tr>
			<td>Biofuge Stratos high-speed refrigeration centrifuge</td>
			<td>HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VCX130 ultrasonic fracturing instrument</td>
			<td>SONICS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACS-ZEAS electronic scale</td>
			<td>Phos technology development, Beijing.</td>
			<td></td>
			<td>The weight of the weighing rats can be accurate to 0.1g.</td>
		</tr>
		<tr>
			<td>Open Field Box</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>wooden box of open field&#160; 100 cm by 100 cm x 40 cm, inside wall and bottom as the gray.The bottom is divided into 25 equal area squares, each of which is 20cm x 20cm, and the 16 grids along the outer wall are the external ones, and the other 9 grids are central.The camera is mounted above the median.</td>
		</tr>
		<tr>
			<td>Elevated Plus-maze</td>
			<td>Beijing zhongshi dechuang technology development co. LTD.</td>
			<td></td>
			<td>The open arms and close&#160; arms of the cross are composed of 30cm x 5cm x 15cm, and the central area is 5cm x 5cm, with a camera mounted above the center and 45cm high.</td>
		</tr>
		<tr>
			<td>rat swimming bucket.</td>
			<td>Zhenhua biological instrument equipment co., LTD. Anhui,China.</td>
			<td></td>
			<td>The volume of plastic drum is 70cm x 30cm x 110cm, which is used for swimming in rats.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Shiya instrument co., LTD., changzhou,China.</td>
			<td></td>
			<td>Control water temperature</td>
		</tr>
		<tr>
			<td>Small water pump</td>
			<td>Xincheng technology co., LTD., chengdu,China.</td>
			<td></td>
			<td>Used for water tank and swimming behavior.</td>
		</tr>
		<tr>
			<td>Ethovition3.0 behavioral software.</td>
			<td>Nuldus,Netherlands</td>
			<td></td>
			<td>Measurement analysis of rat behavior videos.</td>
		</tr>
	</tbody>
</table>

a rat model of central fatigue using a modified multiple platform method

Bu makalede, biz merkezi yorgunluk değiştirilmiş kullanarak bir sıçan model tanıttı birden çok platform yöntemi (MMPM). Birden çok Platform kutusu altındaki dar platformlar bir su haznesi olarak tasarlanmıştır. Modeli fareler tank içine koymak ve platformlar için 14 h (18:00-8:00) 21 gün üst üste için gün başına karşıtlığını ayarla boş kontrol grubu ile durdu. Modelleme sonunda, sıçan modeli grubunda belirgin bir yorgun görünüm gösterdi. Model değerlendirmek için biz birkaç davranış testleri yapılan, dahil olmak üzere açık alan (sık sık), yüksek artı labirent (EPM) test ve ayrıntılı yüzme (ES) testi test. Sonuçlar bu anksiyete, mekansal biliş bozukluğu, zavallı kas performansı ve reddedilen gönüllü etkinlik modeli fareler içinde sunulan Tanı Merkezi yorgunluk onaylamak gösterdi. Merkezi nörotransmitter değişiklikleri de sonuç doğrulandı. Sonuç olarak, model başarıyla merkezi yorgunluk benzetimli ve gelecekteki çalışma modeli ile hastalığın patolojik mekanizması açığa yardımcı.

Biz merkezi yorgunluk MMPM kullanarak bir fare modeli açıklar. 24 Wistar fareler rasgele kontrol grubu ve her grupta 12 farelerle model grubu ayrılır. Modeli aparatı (Şekil 1) altta dar platformlar bir su haznesi olarak tasarlanmıştır. Modeli fareler için 21 gün (Şekil 2) kısmi uyku süresi dahil olmak üzere günde 14 saat platformlarda stand.
Davranış t...

Watch this Scientific Journal Video about A Rat Model of Central Fatigue Using a Modified Multiple Platform Method at JoVE.com

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Burada, merkezi yorgunluk değiştirilmiş kullanarak bir fare modeli tanıtmak için bir protokol mevcut birden çok platform yöntemi (MMPM).

Bir fare modeli değiştirilmiş bir kullanarak merkezi yorgunluk birden çok Platform yöntemi

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

a-rat-model-central-fatigue-using-modified-multiple-platform

Research

JoVE Journal

Neuroscience

8.9K Görüntüleme.  Beijing University of Chinese Medicine. Burada, merkezi yorgunluk değiştirilmiş kullanarak bir fare modeli tanıtmak için bir protokol mevcut birden çok platform yöntemi (MMPM).

Video: A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Dinamik Kelepçe Tekniği ile Sıçan orta beyin dopaminerjik nöronlar NMDA Reseptör İletkenlik Uygulama

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Nörobilim

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Regioseçici Biolistik Modifiye Gen Tabancası kullanarak Organotipik Beyin Dilimleri Hedefleme

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Sprague-Dawley Sıçan nodoz Ganglionlar Enjeksiyon Bir Yöntem

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Kayıt ve Stimülasyon Uygulamaları için Yüksek İletişim-Yoğunluk Fabrikasyon, Düz Arayüz Sinir Elektrotlar

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Dinamik Kantitatif Duyusal Test Merkezi Ağrı İşleme karakterize etmek

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

Multipl Skleroz Fare Modeli Progressive Nörolojik Engellilik Ölçümü

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subaraknoid kanama, bir fare modeli serebral Vasospasm miktar hacimsel bir yöntemi

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Birden fazla Motor yolları sıçan Spinal Kord Lentiviral bir vektör direkt enjeksiyon vurgulamaktadır

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Murine glia'nın Multipl Merkezi Sinir Sistemi Bölgelerinden Diseksiyonu ve İzolasyonu

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Sıçan Beyin Dokusu için Hidrofobik Doku Temizleme Yöntemi

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...