Bu çalışmada, FMMU yeni bir temel ve kısmen NIH finansman bulma desteklenmiştir.

1. Glia Hücre Kültür Glial hücreleri, merkezi sinir sisteminin farklı bölgelerinde yetiştirilebilir. Tüm süreç süreç resimde gösterilmiştir. <img src="/files/ftp_upload/2111/2111process.jpg" alt="Süreç 1" /> Gün 1 Kaplama kültür plaka ve lamelleri <ol><li> Kuru steril cam mikroskop yuvarlak bir otoklavda lamelleri. </li><li> Sterilize 24-kuyu kültür plaka içine Plaka sterilize lamelleri. </li><li> Kat poli-lisin ve kök sıcaklık altında 2 saat süreyle inkübe lamelleri. </li><li> 3. adımı aynı şekilde Kat 6 kuyu kültür plakalar. </li><li> Lamelleri ve 6-iyi bir kültür plaka iki kez distile su ve kültür kaputun içindeki kuru hava ile yıkayın. </li><li> 37 derece altında,% 5 CO 2 ile 6-plaka 24-plaka ve 2 mL her bir kuyu içinde her bir kuyunun 200 mcL DMEM orta (% 10 FBS) ekleyin ve kuvöz içine koydu </li></ol> 2. Gün Yalıtımlı korteks ve glial hücre kültürü <ol><li> Pozitif akış diseksiyonu kaput sterilize edin. </li><li> 20 dakika UV ışığı açın. </li><li> % 70 etanol ile tüm yüzeylerde Sprey ve kullanmadan önce 15 dakika bekleyin. </li><li> gergin değiştirmek: anestezisi sıçan hipotermi yavrular (P2-P6) ve işletim makas kullanarak framen magnum üssünde başını kesmek. </li><li> Kafatası kapalı bir iris makas ve soyma kullanarak sagital sütür boyunca kafatası açın. </li><li> Önbeyin çıkarın ve stereomikroskop altında, L15, orta soğutulmuş içine koymak, dikkatli bir şekilde, kan damarları ile dura ve pia membran temiz korteks izole ve L15 orta birkaç kez yıkayın. </li><li> Korteks mikrocerrahi makas ile küçük parçalar halinde kesin. </li><li> Tripsin-EDTA için 37 derece 15 dakika, korteks parçaları ve inkübe içine orta L15 ile hazırlanan% 0.125 ekleyin. </li><li> Final konsantrasyon 10ug/mL DNaz stok solüsyonu eklemek için, doku çözüm aspirat ve yangın parlatılmış cam Pasteure pipet ile 20 kez, tek hücre süspansiyon toplamak, tüp içinde 50ml 20ml DMEM ortama% 20 FBS ile doku blokları transfer. </li><li> DMEM orta, yeniden askıya% 10 FBS ile DMEM hücreleri, 5000-10000/cm 2 hemasitometre, tohum hücreleri ile mikroskop altında hücreleri saymak. Hücre süspansiyonu bir kez yıkayın . 37 derece% 5 inkübatör hücreleri koruyun, orta haftada 2 kez yarım değiştirin. </li></ol> 2. Dorsal kök gangliyon Nöronlar İzolasyon, Kültür ve Arıtma 1. Gün Kültür Malzeme hazırlayın <ol><li> Poli-lizin, yıkama lamelleri ile sterilize lamelleri ile iki kez su ve hava kuru damıtmak Coat, 24 kuyu tabak içine koydu. </li><li> % 2 B27 ve 2.5 sn NGF (50 ng / ml) ile 100μL Neurobasal orta, 37 derece,% 5 CO 2 tabak koyun. </li></ol> 2. Gün embriyolar DRG&#39;ler Isolate <ol><li> Glial hücre kültürü aynı şekilde akışını kaput sterilize edilmiştir. </li><li> Doz aşımı ile CO 2,% 70 etanol ile sterilize karın hamile bir sıçan Euthanize (E15), embriyolar rahim izole edilmiş ve soğutulmuş L15 orta koymak. </li><li> 35mm yemekleri stereomikroskopta ve transfer altında soğutulmuş L15 orta dorsal kök gangliyon Spinal kabloları izole edin. </li><li> Tek hücre süspansiyonu toplayın ve NBF orta (Neurobasal orta% 2 B27 ve 2.5 sn NGF (50 ng / ml) içeren) ile bir kez yıkayın. </li></ol> 3. Gün Purify DRG Nöronlar <ol><li> 18h-24h nöron tohumlama sonra, nihai konsantrasyonu 20 mcM ve son hacim 200 mcL nöron kültürüne 1 mM konsantre FUDR stok solüsyonu ekleyin. </li><li> 72h sonra Neurobasal orta FUDR olmadan% 2 B27 ve 2.5 sn NGF (50ng/mL) içeren yarım orta yerine. Bundan sonra, her gün orta değiştirin. </li></ol> 3. Glial hücreleri ile Coculture DRG nöronlar <ol><li> Glial hücre kültürü izdiham (20 gün sonra tohumlama) ulaştığında, bu nöronlar ile coculture hazır. </li><li> 24 saat arıtılmış nöronlar eklemeden önce, glial hücreler orta,% 2 B27 ve 2.5 sn NGF (50 ng / ml) içeren Neurobasal orta değiştirildi. </li><li> Saf nöronlar kültür kuyulardan toplanmıştır yangın cilalı Pasteure pipet ile mekanik geçerek, tek hücre süspansiyonu elde edildi. </li><li> Nöron sayısını sayma sonra,% 2 B27 ve 2.5 sn NGF (50ng/mL) içeren Neurobasal orta konfluent glial hücreler üzerinde 500/cm 2 numaralı seribaşı. </li><li> Glial hücreler nöronlar yapışma ve neurite büyüme kaydedilmiş ve hücre tipi özel antikorları kullanarak görüntü analiz yazılımı ve immünhistokimya farklı zaman noktalarında analiz olabilir. </li></ol> 4. Temsilcisi Sonuçlar <ol><li> Glial hücre kültürü: tohumlama sonra, glial hücreleri yaklaşık 20 günde konfluent olacak. Faz kontrast microsc altındayerdir, kolayca farklı morfolojik glial hücre alt farklı büyüme paterni altyapılar oluşturdu ve immuocytochemisty tekniği (Şekil 1, Şekil 2) gösterildiği gibi GFAP pozitif astrositler% 90 daha fazla olduğu tespit edilmiştir. </li><li> DRG nöronlar kültürü: yöntem, 72 saat FUDR tedavi sonra, DRG nöronların saflık 6 gün içinde% 99 gibi yüksek ulaşacak, DRG nöronlar kendi benzersiz morfolojileri gösterdi ve yüksek yoğunluklu neurite büyüme (Şekil 3, Şekil 4). </li><li> Glial hücre ve nöronlar coculture: DRG nöronlar yapışma ve neurite akıbet tohumlama sonra 4 saat içinde glial hücreleri kolayca oluştu, dikkatli gözlemler nöron yapışma ve neurite büyüme özel büyüme paterni altyapılardan oluşan glial hücre alt etkilenmiş olduğunu gösterdi, bu kolayca tespit edilebilir faz kontrast mikroskop altında ve immünsitokimya (Şekil 5, Şekil 6). </li></ol><img src="/files/ftp_upload/2111/2111fig1.jpg" alt="Şekil 1" /> Şekil 1. Konfluent glial hücrelerin morfolojisi. Kortikal glial hücreleri polylysine kaplı lamel kaplama ve 20 gün kültüre, glial hücreler farklı büyüme paterni not, sol tarafındaki hücreleri yayılan bir şekilde düzenlenmiştir. <img src="/files/ftp_upload/2111/2111fig2.jpg" alt="Şekil 2" /> Şekil 2. Glial fibriler asidik protein (GFAP) antikor etiketli konfluent glial hücreleri sağ tarafta GFAP (kırmızı) pozitif hücrelerin yayılan düzenleme unutmayın. <img src="/files/ftp_upload/2111/2111fig3.jpg" alt="Şekil 3" /> Şekil 3. Dorsal kök gangliyon nöronları FUDR tedavi olmadan in vitro büyüdü. Kirlenmesine neden olan hücreleri nöronların background DRG kurdu. <img src="/files/ftp_upload/2111/2111fig4.jpg" alt="Şekil 4" /> Şekil 4. Dorsal kök gangliyon nöronları FUDR tedavi sonra in vitro büyüdü. Arka plan kirlenmesine neden olan hücreler tamamen ortadan kaldırılmıştır. <img src="/files/ftp_upload/2111/2111fig5.jpg" alt="Şekil 5" /> Şekil 5. Glial hücreler üzerinde yetişen Dorsal kök gangliyon nöronları. Nöronlar yapışma ve yayılan düzenlenmiş hücreleri inhibe neurite büyüme ve sağ tarafında glial hücreler üzerinde sınırlı kalmıştır. <img src="/files/ftp_upload/2111/2111fig6.jpg" alt="Şekil 6" /> Şekil 6. Nörofilaman antikor etiketli glial hücreler üzerinde büyüyen Dorsal kök gangliyon nöronları neurite (yeşil), yayılan sol tarafında düzenlenen glial hücreleri inhibe ve sağ tarafında sınırlı idi, tüm glial hücreler GFAP antikor (kırmızı) etiketli .

<ol>
	<li>Rossignol, S., Schwab, M., Schwartz, M., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+time+to+move?.">Spinal cord injury: time to move?.</a> J Neurosci. 27 (44), 11782-11792 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7 (8), 617-627 (2006).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Matyash, V., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+astrocyte+morphology+and+physiology.">Heterogeneity in astrocyte morphology and physiology.</a> Brain Res Rev. , (2009).</li><li>Wanner, I. B., Deik, A., Torres, M., Rosendahl, A., Neary, J. T., Lemmon, V. P., Bixby, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+model+of+the+glial+scar+inhibits+axon+growth.+Glia.">A new in vitro model of the glial scar inhibits axon growth. Glia.</a> 56 (15), 1691-1709 (2008).</li><li>Kuffler, D. P., Sosa, I. J., Reyes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+chondroitin+sulfate+proteoglycan+inhibits+dorsal+root+ganglion+neuron+neurite+outgrowth+and+substrate+specificity+via+a+soma+and+not+a+growth+cone+mechanism.">Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.</a> J Neurosci Res. 87 (13), 2863-2871 (2009).</li><li>Kleitman, N., Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+method+for+the+study+of+myelination.">Tissue culture method for the study of myelination.</a> Culturing nerve cells. , P559-P559 (1998).</li></ol>

Bu deney protokolü, glial hücreler, nöron yapışma ve neurite büyüme özellikle astrosit heterojenite etkisi çalışma iki hedeflere ulaşmak için tasarlanmıştır. Ilk golü bu deneyde astrosit heterojenite, mümkün olduğu kadar korumak için, konfluent glial hücre kültürü zenginleştirilmiş astrositler daha fazla hücre hasarı ve astrositler yaralanması tepki neden olabilecek herhangi bir kimyasal tedavi ve sindirim yayılma olmadan birincil kültür karışık oldu. Son astrosit saflık fibrobasts F...

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<td>DMEM(high glucose)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-039</td>
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<td>L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11415-114</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437-077</td>
<td>&nbsp;</td>
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<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
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<td>poly-lysine</td>
<td>&nbsp;</td>
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<td>P4832</td>
<td>culture grade</td>
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<td>NGF(2.5S)</td>
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<td>Invitrogen</td>
<td>13257-019</td>
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<td>Invitrogen</td>
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<td>FUDR</td>
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<td>neurofilament antibody</td>
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study glial cell heterogeneity influence on axon growth using a new coculture method

Tüm memelilerin merkezi sinir sisteminde, yaralanma sonrası kopmuş aksonları 1 çok kötü orijinal hedefleri ve fonksiyonel iyileşme, rejenerasyon mümkün değildir. Akson rejenerasyonu yetmezliği kombine düşmanca glial hücre çevre, miyelin ilgili inhibitör molekülleri ve içsel nöron rejeneratif kapasitesi 2 azalma da dahil olmak üzere çeşitli faktörlerin bir sonucudur. Astrositler merkezi sinir sisteminde en baskın glial hücre tipi ve fizyoloji ve patoloji koşulları 3 altında akson fonksiyonlarında önemli rol oynar. Kontrast, homolog oligodendrosit astrositler farklı morfolojileri ve gen ekspresyonu 4 farklı astrosit alt popülasyonlar oluşan heterojen bir hücre popülasyonu vardır. Akson büyüme üzerindeki etkiler bu heterojenite fonksiyonel önemi, büyük ölçüde bilinmemektedir. Özellikle glial hücre, nöron davranış astrosit heterojenite fonksiyonunu incelemek için, biz, ortak kültür, sıçan korteks elde glial hücreleri ile yüksek saflaştırılmış dorsal kök gangliyon nöronları yeni bir yöntem oluşturdu. Bu teknikle, aynı koşul altında farklı astrositler alt popülasyonlar nöron yapışma ve akson büyüme karşılaştırabileceksiniz. Bu raporda, astrositler izolasyonu ve kültürü, dorsal kök ganglionlar nöronlar izolasyon ve saflaştırma ve astrositler ile nöronların DRG ko-kültür için bu yöntemi ayrıntılı protokol verir. Bu yöntem ayrıca, nöronlar ve glial hücreler arasındaki hücresel veya bölgesel belirli bir etkileşim çalışması için diğer beyin bölgelerine uzatılabilir.

Watch this Scientific Journal Video about Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method at JoVE.com

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Bu protokol, glial hücre heterojenite bir akson büyüme üzerindeki etkisini incelemek için yeni bir yöntem tarif<em&gt; In vitro</em&gt; Ko-kültür sistemi. Rat kortikal glial hücreleri birleştiği kültürlü ve yüksek sıçan dorsal kök gangliyon nöronları saflaştırılmış ile kültüre edildi. Farklı glial hücre nöronlar yapışma ve akson büyüme etkisi aynı kültürü doğrudan karşılaştırıldı. Bu yöntem, doğrudan nöron yapışma ve akson büyüme glial hücre heterojenite etkisini incelemek için yeni bir yol sağlar.

Axon Büyüme, Yeni Bir Coculture Yöntemle Çalışması Glial Hücre heterojenite Etkisi

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

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Neuroscience

17.2K Görüntüleme.  Cedars Sinai Medical Center, UCLA. Bu protokol, glial hücre heterojenite bir akson büyüme üzerindeki etkisini incelemek için yeni bir yöntem tarif In vitro Ko-kültür sistemi. Rat kortikal glial hücreleri birleştiği kültürlü ve yüksek sıçan dorsal kök gangliyon nöronları saflaştırılmış ile kültüre edildi. Farklı glial hücre nöronlar yapışma ve akson büyüme etkisi aynı kültürü doğrudan karşılaştırıldı. Bu yöntem, doğrudan nöron yapışma ve akson...

Video: Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Kültür Yetişkin Dorsal Root Ganglion Nöronlar ile Axon Rejenerasyon Genetik Çalışma

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Nörobilim

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Bir<em&gt; Caenorhabditis elegans</emAmylopathy Çalışmaları&gt; Model Sistemi

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

Yenidoğan Kemirgenlerin Ventral Kafatası kranyotomi olun Bir Yöntem

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Nöronal Ateşleme Oranlarının Manipülasyon tarafından Astrositik reseptörleri Plastisite Inducing

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Yetişkin Fare Omurilik Doku İlköğretim Karışık glial Kültürler hazırlanması

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Doğrusal Agaroz Kanallar kullanarak Okuyacak<em&gt; Drosophila</em&gt; Larva Tarama Davranış

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

Tatlandırıcı Kodlamanın Yeni Yöntemleri

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Görselleştirme Motor Axon navigasyon ve hafif levha Floresans mikroskobu kullanarak fare embriyo Axon Arborization miktar

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optik koherens tomografi ve Optokinetic yanıt yapısal ve işlevsel görsel sistemin çıktıları fareler ve sıçanlar olarak kullanma

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Glial ve Nöronal Hücrelerin 3D Rekonstrüksiyonu ve Sanal Gerçeklik Analizi Için Bir Yöntem

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...