- Pedro Barrie de la Maza Vakfı bu çalışmaları destekledi.

1) Rotenone intragastrik İdaresi <ol><li> Hayvan tartılır. </li><li> Gerekli rotenone / araç çözüm toplam hacmi hesaplayın. Bu durumda hayvan ağırlığı gram başına 0,01 ml. </li><li> Önceden hesaplanmış rotenone çözüm hacmi (0.625 mg / ml rotenone,% 4 karboksimetilselüloz ve% 1.25 kloroform) veya araç çözüm (% 4 karboksimetilselüloz ve kloroform% 1.25) ile 1 ml şırınga şarj edin. Bu durumda, bir 5 mg / kg rotenone doz karşılık gelir. </li><li> Öte yandan ilk iki parmakları ile çekerek fare Pick up ve fare kuyruğu, tek elle tutarak, boyun cildi germek. </li><li> Başı iki parmak arasında sabit bir kez, elin avuç içi karşı yalın ve baş aşağı çevirin. Dördüncü ve beşinci parmakları kullanarak kuyruk sabitleyin. </li><li> Baş arkaya doğru tutmak, damak farenin ağzına açmak için tuşuna basın. Yavaşça ağzına gavaj tanıtmak. Sonda bir iğne ve düzenli bir iğne kullanmak önemlidir. Yavaş yavaş, yeteri kadar mide yönde sonda taşımak. Bu akciğerlerin elde malzeme önlemek olacaktır. </li><li> Emboli hafifçe basarak rotenone çözüm veya araç yönetin. Tamamlandığında, yavaş yavaş gavaj ayıklayın. </li><li> Bütün hayvanlar bir kafes yapılır kadar başka bir kafeste hayvan geri yerleştirin. </li></ol> 2) immün Prosedürleri <ol><li> Tedaviden sonra, fare Ketamin 100 mg / kg und Xylazin 10 mg / kg ip ve intrakardiyak PBS içinde% 4 Paraformaldehit ile perfüze kullanarak anestezi. </li><li> Cesaret sonra diseksiyon tarafından çıkarılan ve bir gecede% 15 sakaroz yerleştirilir. Ertesi gün, doku% 30 sakaroz aktarılır ve 4 gecede tekrar inkübe ° C </li><li> Daha sonra doku Doku tek yerleştirilir ve kullanana kadar -80 ° C derin dondurucuda saklanır. </li><li> Bir Kriyostat kullanarak, 20 mikron bağırsak kesitleri SuperFrost slaytlar aktarılır. </li><li> Anti-βIII-tubulin ve anti-alfa-sinüklein antikorları kullanarak prosedürleri Engelleme ve immün sonra daha önce açıklanan 9 olarak yapılmaktadır. </li></ol> 3) Alfa-sinüklein İçerme Görüntü Analizi Bu görüntüleri &#39;kökenli habersiz harici bir kişi analizi gerçekleştiren tavsiye edilir. Bu nedenle, veri kodlanmış olmalıdır. Görüntü analizi Fiji yazılımı kullanılarak yapıldı. <ol><li> Konfokal görüntü dosyasını açın. Daha sonra, bu her kanal bağımsız çalışmak mümkün olduğunu kanalları ayrıldı. </li><li> Bu kanalların kullanımı ve Analiz&gt; Ölçek ayarlama ve 1 Piksel boyutu ayarlamak seçmek için gidiş değildir kapatın. 0,13 (bu amacı ve kullanılan çözünürlük bağlıdır) olarak bilinen mesafeyi ayarlayın ve küresel tıklayın. Görüntünün boyutu artık mikron gösterilir. </li><li> Alfa-sinüklein kanalı seçin ve ganglion seçin, seçimi tüm dilimleri ganglion kapsadığından emin olun. </li><li> Basın Image&gt; görüntü çoğaltmak için çoğaltın. Ganglion seçildiğinde, sadece bir görüntü boyutu ganglion yinelenir. </li><li> Basın Düzenle&gt; Temizle dışında. Seçilen bölge dışından görüntü parçası siyah olacak. </li><li> Prosedürü ne kadar iyi gitti sonraki adımları belirlemek için görüntüyü yeniden çoğaltın. Gürültü arka plan oranı yüksek bir sinyal Görüntüler görüntü analizi için kullanılan olmamalıdır. </li><li> Image&gt; Adjust&gt; Threshold seçin ve sonra MaxEntropy seçin. Sonra prosedürü iyi gittiğini sağlamak için kırpılmış resim ve özgün bir dilimleri üzerinde taşıyın. Entropik eşik görüntü histogram ve bu eşiğin daha yüksek şiddetlerde siyah resmin üzerine beyaz görünecektir, yalnızca piksel dayalı bir yoğunluk sinyal seviyesi seçer. </li><li> Süreç seçin, ardından Pürüzsüz tıklayın. Bu adım pixeled kenarları düzgün kenarlara görüntüleri dönüştürür. </li><li> Süreç seçin, sonra İkili tıklatın ve İkili olun seçin. Görüntü üzerine siyah beyaz görüntü olarak görünecektir. </li><li> Çoğaltılmış görüntü sonraki adımı gerçekleştirmek için görüntü çoğaltın. </li><li> Analiz&gt; analiz parçacıklar üzerine tıklayın. Sonra Göster&#39;i seçin: Anahatları ve özetleyin tıklayın. Varsayılan ayarları geri kalanı yapraklı olmalıdır. Bu fonksiyon, bir sinyal ve birlikte gruplandırılır toplam piksel sayısı tanır. Sinyal artı piksel gruplarının sayısı ve ortalama büyüklüğü piksel toplam yüzey rapor edilmiştir. </li><li> Karşılaştırın ve iyi ayarlayan büyük dilim seçin. Dilim işaretlenir. </li><li> Toplam alfa-sinüklein yüzey kapanımlar sayısı ve ortalama partikül boyutu ile veri tablosu kopyalayın. Verileri bir excel tablo ya da başka bir yerde açıklamalı içine yapıştırın. </li><li> Fiji dönün ve β-tubulin kanal önceden seçilmiş bir dilim bulmak ve seçmek. </li><li> Ganglion alanı seçin ve sonra ölçün seçin analiz. Başka bir tablo gösteren görünecektirganglion toplam yüzey. </li><li> Alfa-sinüklein ölçüm yakın excel sayfasına tablodan veri ayıklamak ya da başka bir yere kopyalayın. </li><li> Excel kullanarak, alfa-sinüklein ölçüm ganglion boyutuna göre normalize toplam alfa-sinüklein yüzey ve içerme numarası ganglion yüzey elde etmek için elde edilen farklı parametreler bölün. </li></ol> 4) Temsilcisi Sonuçlar Gavaj yönetim protokolü doğru yapıldığında sakıncaya hayvan için en az düzeydedir. Tedavi rotenone bu konsantrasyonu kullanılarak kan rotenone düzeyleri olmadan ENS üzerinde rotenone yerel bir etkisi sağlar ve kas veya beyin mitokondriyal Kompleksi inhibisyonu ben bile tedavi 1.5 ay sonra (bkz. Şekil 1). Görüntü analiz doğru yapılmazsa, alfa-sinüklein desen titiz bir analiz mümkün olmalıdır. Alfa-sinüklein (toplam yüzey), alfa-sinüklein hücre deseni (inklüzyon sayısı) ve alfa-sinüklein inklüzyon varlığı (dahil boyutu) (Şekil 2) toplam tutarı hakkında güvenilir veriler verir. <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="Şekil 1" /> Şekil 1. Rotenone Motor fonksiyon bozukluğu, kan ya da MSS rotenone algılama olmadan fareler davrandı. A, standart (50 ng / ml) ve 20, 10 ve 5 mg / kg tedavi edilen farelerin beyin örnekleri kromatogram B ve C, ölçümü, rotenone kan düzeyleri (B) ve MSS (C) B, kan seviyeleri 1 , tedaviden sonra 2 ve 3 saat. Fareler üç grup (n = 3) bölünmüş ve 2.5, 5, 10 ve 20 mg / kg rotenone ile tedavi edildi (n = 3), 300 mcL kan rotenone uygulamadan sonra 1, 2 ve 3 saat ayıklanır ve bir araya toplanmış oldu HPLC analizi ile günde 5 (n = 3), 10 (n = 3) ve 20 mg / kg (n = 1) rotenone, beyin ve beyin sapı 2 saat sonra 1 çıkarılan ve bir kez C, fareler bir hafta süreyle tedavi edildi son yönetim ve HPLC analizi D, mitokondriyal Kompleksi 1,5 ay tedavi edilen farelerde kas ve beyin örnekleri faaliyet için hazır. <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="Şekil 2" /> Şekil 2. Yerel idare rotenone ENS ganglionlarda gliozis ile alfa-sinüklein fosforilasyon, birikim ve toplama indükler (20 mikron ölçekli barlar). A, B, C, anti βIII-tubulin, alfa-sinüklein ve onikiparmak bağırsağı DAPI boyama (B) ve ileum ( A, C) bölümleri. 3 ay kontrolleri (A) kıyasla B Ok, 1.5 ay tedavi, enterik sinir sistemi ganglionlar içinde artan bir alfa-sinüklein noktalı desen kaynaklı . C Ok, büyük alfa-sinüklein kapanımlar (Ø&gt; 6 mm) 3 ay tedavi indüklenen oluşumu D anti-alfa-sinüklein, Thioflavine G ve DAPI kullanarak immünofloresan boyama. D Ok, tedavi edilen farelerin sadece 3 ay bu büyük alfa-sinüklein birikimleri agregasyon gösterdi. D, E, D&#39;&#39;, görüntü analiz kantifikasyon adımları, E, bir ENS ganglion gösteren nicellendirmesinde kullanılan görüntü örnek gösteren alfa sinüklein karşı immunohistokimyasal farklı kapanımlar E &#39;, görüntü analiz protokolü sonra elde edilen görüntü örneği E&#39;&#39;, bazı orijinal kapanımlar (yeşil) ve protokolü (sarı hat) gerçekleştirdikten sonra elde edilen temsili arasındaki örtüşme. FG, gösterilen deney ölçümü AC otomatik segmentasyon ve entropi tabanlı eşikleme yöntemleri kullanılarak yapıldı. Tek yıldız, p &lt;0.05, ve çift-yıldız, p &lt;0.01 F, her sütun toplam alfa-sinüklein yüzey / ganglion yüzey temsil G, her sütun, alfa-sinüklein kapanımlar / ganglion yüzey toplam sayısını temsil eder . Tüm grafikler ortalama ± sem

<ol>
	<li>Braak, H., de Vos, R. A., Bohl, J., Del Tredici, K., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+alpha-synuclein+immunoreactive+inclusions+in+Meissner's+and+Auerbach's+plexuses+in+cases+staged+for+Parkinson's+disease-related+brain+pathology.">Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.</a> Neurosci Lett. 396, 67-72 (2006).</li><li>Wakabayashi, K., Takahashi, H., Ohama, E., Ikuta, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+an+immunohistochemical+study+of+Lewy+body-containing+neurons+in+the+enteric+nervous+system.">Parkinson's disease: an immunohistochemical study of Lewy body-containing neurons in the enteric nervous system.</a> Acta Neuropathol. 79, 581-583 (1990).</li><li>Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+occurrence+of+Lewy+bodies+in+the+dorsal+vagal+nucleus+in+a+patient+with+late-onset+parkinsonism.">Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.</a> J Neurol Sci. 165, 188-191 (1999).</li><li>Braak, H., Sastre, M., Bohl, J. 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Francisco Pan-Montojo bu hayvan modeli (Application sayısı PCT / EP 2009/005688) için bekleyen bir patent başvurusu vardır.

Intragastrik yönetiminin daha önce 12 yapılmıştır . Ancak,% 0.5 karboksimetilselüloz sodyum tuzu (CMSS) tek başına Inden Kullanıcı el yazmasına göre, rotenone verildi çözülmüş. Rotenone yüksek bir liphophylic madde. Bu nedenle, rotenone yalnız% 0.5 CMSS içinde çözünmüş olamaz ve eğer bunu çöktürmek olacaktır. Kloroform yağış kaçınarak eşit olarak dağıtılmış bir rotenone süspansiyon oluşturur. Ayrıca, yüksek konsantrasyonlarda CMSS pestisit nedeniyle, uygulanan ç?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>R8875</td>
<td>Germany</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3313.1</td>
<td>Germany</td>
</tr>
<tr>
<td>Carboxymethylcellulose</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>C5678</td>
<td>Germany</td>
</tr>
<tr>
<td>Gavage 1,2 mm x 60 mm</td>
<td>&nbsp;</td>
<td>Unimed</td>
<td>&nbsp;</td>
<td>Switzerland</td>
</tr>
<tr>
<td>LSM 510</td>
<td>&nbsp;</td>
<td>Zeiss Microscopes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FIJI</td>
<td>&nbsp;</td>
<td>http://pacific.mpi-cbg.de</td>
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oral administration of rotenone using a gavage and image analysis of alpha-synuclein inclusions in the enteric nervous system

Parkinson hastalığında (PH) olan hastalarda, ilgili patoloji, diğerlerinin yanı sıra enterik sinir sistemi (ENS) 1,2, koku ampul (OB), vagus (DMV) 3 dorsal motor çekirdeği, intermediolateral içeren bir karakteristik bir yol izler omurilik 4 ve substantia nigra çekirdeği, hastalığın 4,5 nöropatolojik evreleme için bir temel sağlamaktadır. ENS ve OB en çok maruz kalan sinir yapıları ve ilk etkilenecek olanlar. İlginçtir, PD pestisit maruziyetinin 6-8 ile ilgili olmuştur. Burada ayrıntılı olarak, önceki çalışmada 9 kullanılan iki yöntem gösteriyor . ENS üzerinde yerel olarak hareket rotenone etkilerini analiz etmek için, biz, bir yıl eski C57/BL6 fareler bir sonda kullanarak rotenone yönetilmektedir. Rotenone güçlü mitokondriyal Kompleksi Ben 10 inhibe yaygın olarak kullanılan pestisit. Bu yüksek lipofilik ve kötü gastrointestinal sistem 11 emilir. Bizim sonuçlarımız, 5 mg / rotenone kg yönetimi mitokondriyal Kompleksi kas veya beyin aktivitesi inhibe etmediğini gösterdi. Böylece, bizim yönetim yöntemi rotenone kullanarak hepatoportal sistemi çapraz vermedi ve sadece ENS üzerinde hareket ediyordu düşündüren. Burada pestisitler ENS alfa-sinüklein birikimi pestisit etkilerini analiz etmek için kullanılan bir sonda ve görüntü analizi protokolü kullanarak yönetmek için bir yöntem. Ilk bölümünde istenen hassas konsantrasyonda pestisitler (rotenone) intragastrik yönetimi sağlayan bir yöntem gösterir. İkinci yöntem, bir görüntü analiz yazılımı kullanarak ENS alfa-sinüklein birikimi analiz etmek için bir yarı-otomatik görüntü analiz protokolünü gösterir.

Watch this Scientific Journal Video about Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System at JoVE.com

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Parkinson hastalığı, pestisitlere maruz kalma ile ilgili olmuştur. Burada bir gastrik tüp istenen konsantrasyon ve enterik sinir sistemi alfa-sinüklein birikimi etkilerini analiz etmek için bir yöntem kullanarak pestisitler sağlamak için bir yöntem gösteriyor.

Enterik Sinir Sistemi Alpha-sinüklein İçermelerin bir sonda ve Görüntü Analizi kullanarak Rotenone Oral İdaresi

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

oral-administration-rotenone-using-gavage-image-analysis-alpha

Research

JoVE Journal

Neuroscience

16.9K Görüntüleme.  Technische Universität Dresden. Parkinson hastalığı, pestisitlere maruz kalma ile ilgili olmuştur. Burada bir gastrik tüp istenen konsantrasyon ve enterik sinir sistemi alfa-sinüklein birikimi etkilerini analiz etmek için bir yöntem kullanarak pestisitler sağlamak için bir yöntem gösteriyor.

Video: Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Nörobilim

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Kemirgen Merkezi Sinir Sistemi İlaçların doğrudan Intraventriküler teslim

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

Kemirgen Santral Sinir Sistemi vivo optogenetic Stimülasyon içinde

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Enterik Sinir Sistemi Yerinde Ca 2+ Görüntüleme

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Parkinson Brains gelen Çözünür ve çözünmez alfa-sinüklein Sıralı Ekstraksiyon

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

Alfa-sinüklein fibrillerinin Çevresel Aşılama sonrası Tranjenik Farelerde Nöroinflamasyondan Biyoparlaklık Görüntüleme

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Yöntemleri ve merkezi sinir sistemi iletim değerlendirilmesi ve Adeno ilişkili virüs fareler için intravenöz Yönetim için ipuçları

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Microsomes ilişkili Alpha-synuclein toplamları için birincil nöronlar eksojen yönetim olarak güçlü hücre modeli liflerinde oluşumu

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Erken Doğum Sonrası Fare Beyinlerine Kemogenetik Mühendislik Kortikal İnkornöron Progenitorlarının Nakli

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Monomers ve Use In vivo Alfa-Synuclein önceden biçimlendirilmiş fibrils üretimi

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...