Biz BAC GLT1 eGFP fareler ve GLT1 antikor sağlamak için Dr Jeffrey Rothstein teşekkür etmek istiyorum; değerli çekirdek imkanları sağlamak için; Sinirbilim Araştırmaları Tufts Merkezi (PI, Rob Jackson NIH P30 NS047243); Yeni öğretim üye hibesi (NIH P30 5P30NS069254-02 ; Tufts Sinirbilim Anabilim PI, Phil Haydon).

1. Mikroakışkan Kültür Odası Meclisi (MCP) <ol><li> MCP (Şekil 1) hücreleri, 4 farklı bölmeli kültür için tasarlanan açık odaları vardır. Tipik olarak, merkezi kanal (çapı 3 um) ile bağlanmış iki bölmeden oluşmaktadır. Cam tabanlı yemekleri ile MCP Meclisi kültür ve sonraki görüntü analiz hazırlamak için gereklidir. </li><li> İlk olarak, Polyornithine (Sigma-Aldrich, 1 mg / ml) ile kat steril cam tabanlı bir bulaşık sodyum tetraborat (Sigma-Aldrich, 10 mM pH 8.5) içinde çözüldü ve 37 ° C de bir gece boyunca inkübe edildi </li><li> Ertesi gün, kaplanmış cam tabanlı bir bulaşık GKD 2 O ile üç kez yıkandı ve steril davlumbaz altında kurutulmuştur. Cam tabanlı tabaklar daha sonra daha fazla laminin (Sigma-Aldrich, 1 mg / ml) ile kaplanır, 1 saat için UV ışık altında kurutulmuştur. Kaplanmış bulaşık kullanıma hazır veya kullanıma dek -20 ° C&#39;de muhafaza edilebilir. Bu kaplamaco-kültür kaplama nöronların ve astrositler için gerekli yeniden. </li><li> Kültür platformu montajı için, mikroakışkan kültür platformları sıkı bir mühür oluşturmak için onun alt tarafında merkezi bağlayan kanalları ile kaplı cam zeminli yemeklerin üstüne yerleştirildi. Normal nöron veya astrosit kültür ortamlarda monte MCP her iki tarafı da (Şekil 1) (hücre kaplama alanlarından) eklendi ve 37 ° C&#39;de 2-4 saat inkübe, MCP ve cam arasında hiçbir sızıntı olmadığından emin olmak için yapıldı dipli çanak. Biz hiçbir sızıntı olmadığından emin olmak için kaplama hücreleri önce orta ile test ettik. Cam dipli çanak MCP montajı taze kullanılmadan önce hazırlanmalıdır. </li></ol> 2. Montajlı MCP nöronal Aksonlar nöronal Kültür ve İndüksiyon hazırlanması <ol><li> Kortikal nöronal hücre kültürleri taze embriyonik 14-16 günlük eski fare beyninden hazırlanmıştır. Nöron kültür ortamı neurobasal ortamı,% 2 B27 neurobasa oluşmaktadırl takviyesi, 100x GlutaMAX% 1 ve% 1 penisilin-streptomysin ekleyerek glutamin 2 mM. Fare beyin disseksiyon takiben, zarları mikroskop altında korteks çıkarıldı. Doku daha sonra tripsin ile maruz kalan yüzey alanını arttırmak için küçük doku blokları için bir jilet ile kıyılmış edildi. Doku blokları 37 ° C su banyosu içinde 10 dakika için (Sigma-Aldrich,% 0.05 tripsin) tripsinize ve bir ateş cilalı Pastör pipeti ile tritürasyon ile ayrılmış ve sonra yavaşça edildi. Disosiye berrak nöronal hücrelerin hücre süspansiyonu toplamak için bir 70 mikron filtre içinden filtre edildi. </li><li> Nöronların hücre tutma alanı (Şekil 1) bağlamak ve böylece nöronların (2-3 x 10 6 / ml, 150 ul) toplandı MCP sağ tarafında (Şekil 1) hücre kaplama alanı üzerine ekildi. Taze hazırlanmış nöronlar düzenlemeler içine% 5 fetal sığır serumu (Hyclone) ile desteklenmiş nöron orta kaplama ile kaplanmıştırr nöron kültür ortamı. Hücre tutma alanında takmak sadece nöronlar merkezi kanaldan monte MCP diğer tarafına aksonlar büyümesi için gerekli olduğundan, yeterli nöron bağlı olan sağlamak için hücre kaplama alanında nöron plaka yüksek yoğunluk önemlidir hücre saklama alanı. Nöronal aksonların yüksek yoğunluklu bir temsilcisi, görüntü Şekil 2A&#39;da gösterilmiştir. </li><li> Ertesi gün, nöron kaplama orta düzenli nöron kültür ortamı ile değiştirildi. Değişen ortam dikkatlice odasının hücre kaplama alanından orta aspirasyon ile gerçekleştirilir, ancak monte MCP hücre tutma alanı, hücre tutma alanı ve merkezi bağlantı kanalı içinde hava kabarcıkları önlemek için. Edildi Hava kabarcıkları ciddi MCP merkez kanal içine akson akıbet ve izleyen girdi inhibe olacaktır. Glial-hücresi kaynaklı nörotrofik faktör (GDNF, 10-20 ng / ml), aynı zamanda, diğer (sol) tarafında eklendimonte MCP (Şekil 2A) merkez kanalları aracılığıyla nöronal yan ve çapraz akson akıbet 5 indüksiyonu kolaylaştırmak için aynı gün odasından. GDNF olmadan spontan akson uzantısı rayiç miktarı da monte MCP merkezi kanallardan nöronal yan ve çapraz görülmektedir. Merkez kanal içine girmek Aksonlar genellikle 2-3 gün sonra kaplama görülür. Aksonlar merkez kanal içine girdikten sonra, genellikle bir gün içinde diğer tarafı girin. Aksonlar diğer yan girdikten sonra, en az bir hafta süreyle normal olarak sağlamdır. </li></ol> 3. Bir Bölümlere Co-kültür Sistemi Kurmak MCP için Kültürlü astrositlerin eklenmesi Primer astrosit kültürleri, P1-3 pup fare beyninden hazırlandı. Beyin ayrışma işlem yukarıda tarif edilen nöronal hücre izolasyon prosedürü ile aynıdır. Astrositler ilk ön edildi 10 cm çanak içine ekildiPolyornithine (Sigma-Aldrich, 1 mg / ml) ile kaplanmıştır. Astrosit besiyeri (DMEM,% 10 fetal sığır serumu,% 1 penisilin-streptomysin) enkaz kaldırmak için önümüzdeki iki gün boyunca her gün değiştirildi. Bundan sonra, ortam her üç günde bir değiştirildi. <ol><li> Astrositler 90% konfluent haline ve 7 gün sonra bir tek tabaka oluştururlar. Konfluent astrositler tripsinize edildi ve akson yaklaşık girmek için olan ya da sadece sol tarafında içine girdikten sonra yeniden askıya alınmış astrositler (1x10 6 / ml) 150 ul monte MCP sol tarafında hücre kaplama alanı içine yeniden ekildi MCP toplandı. Nöronlar kaplama vardı sonra astrositler genellikle 4-5 gün ekildi. GDNF birinci MCP sol tarafına astrositlerin yeniden kaplama önce çıkarıldı. Yeniden kaplama astrositler gibi immun GFAP (Şekil 1) gösterildiği gibi, hücre tutma bölgesinde bağlanmıştır. Odacıkların her iki tarafı arasında orta sadece asgari akım (d floresan ile belirlenirDaha önce 4,6 gösterildiği gibi evet), MCP bulundu. </li><li> Astrositler 90% konfluent haline ve 7 gün sonra bir tek tabaka oluştururlar. Konfluent astrositler aksonlar yaklaşık girmek için veya sadece sol içine girmiş olduğu zaman monte MCP sol tarafında hücre kaplama alanı içine yeniden ekildi tripsinize ve yeniden asılı astrositler (1 x 10 6 / ml) 150 ul edildi monte MCP tarafı. Nöronlar kaplama vardı sonra astrositler genellikle 4-5 gün ekildi. GDNF birinci MCP sol tarafına astrositlerin yeniden kaplama önce çıkarıldı. Yeniden kaplama astrositler gibi immun GFAP (Şekil 1) gösterildiği gibi, hücre tutma bölgesinde bağlanmıştır. Daha önce gösterildiği gibi, 4,6 odacıkların her iki tarafı arasında orta sadece asgari akım (floresan boyalar ile belirlenen), MCP bulunmuştur. </li><li> Astrositlerin yeniden kaplama sonra, bir araya MCP sol tarafında girilen aksonların doğrudan doğruya astr ile etkileşimeDoğrudan aksonal temas veya çözünebilir faktörlerin salınımını biri tarafından ocytes. Astroglial plazma membranı glutamat taşıyıcı GLT1 ve nöronal βIII-tubulin immün Şekil 2D-F gösterildiği gibi, aksonların ve astrositler arasındaki etkileşimi görselleştirmek için yapıldı. </li></ol>

<ol>
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Çıkar çatışması ilan etti.

MCP tabanlı nöron ve astrosit ko-kültür sistemi sadece aksonlar merkez kanalları geçmesine izin ve Astroglial hücreleri ile etkileşerek astroglial sinyal yolları ayrıntılı nöronun diseksiyon sağlar. Bu ko-kültür sistemi rahatlıkla konvansiyonel nöron ve astrosit kültürü prosedürleri ile kurulabilir. Biz de Astrositlerde akson bağımlı GLT1 organizatörü aktivasyonu gösteren bir eGFP tabanlı muhabiri istihdam ederek bu ko-kültür sistemi pratik bir uygulama nitelendirdi. <p class="jove_con...

<table border="1"><tbody><tr><td> Fetal bovin serumu </td><td> Hyclone </td><td> SH30070.03 </td><td> nöron cutlure orta nöron kaplama için </td></tr><tr><td> Fetal bovin serumu </td><td> Sigma-Aldrich </td><td> F4135 </td><td> astrosit kültür ortamı için </td></tr><tr><td> Glial türetilen sinir faktörü </td><td> Ar-Ge sistemleri </td><td> 212-GD </td><td> Odanın nöron tarafına 10-20 ng / ml Uygula </td></tr><tr><td> Dulbecco modifiye kartal orta yüksek glukoz </td><td> Sigma-Aldrich </td><td> 11995 </td><td></td></tr><tr><td> 70 mm hücre süzgecinden </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Steril cam alt tabak </td><td> Mattek Corporation, </td><td></td><td></td></tr><tr><td> Mikroakışkan kültür platformları </td><td> Xona Mikroakiskan LLC </td><td> SND150 </td><td></td></tr><tr><td> Kültür plakasının 6 kuyu </td&gt; <td> Cellstar </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> Neuron kültür ortamı <ul><li> Neurobasal orta </li><li> % 2 B27 Neurobasal takviyesi </li><li> % 1 100x GlutaMAX eklenmesi ile 2 mM glutamat </li><li> % 1 Penisilin-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Kaplama hücre Nöron kültür ortamı <ul><li> Neurobasal orta </li><li> % 2 B27 Neurobasal takviyesi </li><li> % 1 100x GlutaMAX eklenmesi ile 2 mM glutamat </li><li> % 1 Penisilin-streptomysin </li><li> % 5 fetal sığır serumu SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> Astrosit kültür ortamı <ul><li> Dulbecco modifiye eagle orta yüksek glukoz </li><li> % 10 fetal bovin serumu F4135 </li><li> % 1 Penisilin-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Mikroakışkan kültür platformu tabanlı nöronal akson ve glia ko-kültür sisteminde kullanılan Tablo 1. Malzemeler. </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

Glia etkileşim Uygun nöron merkezi sinir sistemi (MSS) fizyolojik fonksiyonu için önemlidir. Bu çift yönlü iletişim sophisticatedly nöron ve glia 1,2 arasında belirli sinyal yolakları aracılık etmektedir. Bu sinyal yollarının tanımlanması ve karakterizasyonu glia etkileşim şekilleri MSS fizyolojisi nasıl nöronun anlayışı esastır. Önceleri, nöron ve glia karışık kültürlerin yaygın nöron ve glia arasındaki sinyal yolları test ve karakterizasyonu için kullanılmıştır. In vivo araçları bu müstahzarlar ve diğer öğrendim Ne Ancak, nöron ve glia arasında karşılıklı sinyalleşme sıklıkla nöronlar (yani, akson, dendrit veya soma) 3 içinde belirli bölümlerinde meydana geldiğini öne sürdü. Bu önemli nöronal bölmeleri ayrılması olanak sağlayan yeni bir kültür sistemi geliştirmek için yapar ve özellikle glia ve nöro arasındaki etkileşimi inceliyornal akson / dendrit. Buna ek olarak, geleneksel bir karışık kültür sisteminde çözünür faktörler ve nöron ve glia arasında doğrudan bir kontak sinyali ayırıcı zar yeteneğine sahip değildir. Ayrıca, geleneksel ko-kültür sisteminde nöronların ve glial hücreler ve büyük miktarda, tek bir akson ve bir glial hücre arasındaki etkileşimi gözlemlemek için gerekli çözünürlüğü yoksundur. Bu çalışmada, bir mikroakışkan kültür platformu (MCP) kullanılması ile bir yeni akson ve glia ko-kültür sistemi açıklanmaktadır. Bu ko-kültür sisteminde, nöronlar ve glial hücreler birden santral kanallar aracılığıyla bağlanan iki ayrı odalarında kültüre. Bu mikroakışkan kültür platformu, sadece nöronal süreçleri (özellikle aksonlar) merkezi kanallardan glial tarafında girebilirsiniz. Güçlü floresan protein etiketleme ile birlikte, bu sistem, böyle bir akson / dendrit ve glial etkileşimler arasındaki sinyal yolakları, direkt bakı sağlarnöronal terminalleri glia, glia-aracılı reseptör ticareti, ve glia-aracılı akson büyüme ler akson-aracılı transkripsiyonel düzenleme. Odasının çapı önemli derecede dar akson / dendrit ve glial yüzeyler arasındaki doğrudan zar protein etkileşimi kolaylaştırmak sondalama, glial odasına nöron-zenginleştirilmiş orta akışını engeller.

Astrositlerde akson indüklenen GLT1 organizatörü aktivasyon Time-lapse görüntüleme analizi Bölmeli nöron ve astrosit ko-kültür sistemi sadece nöronal süreçleri, özellikle aksonlar, seçici astrositler ile etkileşim sağlar. Monte MCP içinde akson ve astrosit (veya diğer glial hücreler) ko-kültür başarılı kurulmasını takiben, akson-glia etkileşimleri farklı tür olarak ele alınabilir; Astroglial gen promoter aktivasyonu, astrosit indüklenen akson rehberlik akson ind?...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bu çalışma, bir roman nöronal akson ve (astro) glia ko-kültür platformu kurma prosedürleri açıklar. Bu ko-kültür sisteminde, bir tek akson (ve tek bir glial hücre) arasındaki doğrudan etkileşim manipülasyon glial sinyalleşme karşılıklı nöronların mekanik analiz izin veren, uygun olur.

Mikroakışkan Kültür Platformu Glia Etkileşimi (MCP) tabanlı Nöronal Akson ve Glia Co-kültür Sistemi Nöron görüntülenmesi Analizi

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

imaging-analysis-neuron-to-glia-interaction-microfluidic-culture

Research

JoVE Journal

Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

14.9K Görüntüleme.  Tufts University. Bu çalışma, bir roman nöronal akson ve (astro) glia ko-kültür platformu kurma prosedürleri açıklar. Bu ko-kültür sisteminde, bir tek akson (ve tek bir glial hücre) arasındaki doğrudan etkileşim manipülasyon glial sinyalleşme karşılıklı nöronların mekanik analiz izin veren, uygun olur.

Video: Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Nörobilim

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Mikroakışkan Co-kültür Aygıtları Kullanma Diş Germ innervasyon Geliştirme Analizi

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Dolaylı nöron astrosit Coculture Deneyi: Bir<em&gt; İn Vitro</em&gt; Nöron glia Etkileşimleri Detaylı Soruşturma Set-up

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Yetişkin Sıçan İdrar Kesesinden Primer Nöronların ve Gliaların İzolasyonu ve Kültürü

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Beyin Devrelerinin İnvaziv Olmayan Bağlantısının Hedefli Nöronal Yaralanma

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

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 Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

İskemide Nöron-Glia Etkileşimlerinin Rolünü Nörade İncelemek İçin Hücre Kültürü Modeli

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Fare Kalitesiz Zeytinde Kalsiyum Görüntüleme

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Kalsiyum Görüntüleme Tekniği ile Nöron-Glia Devresindeki Değişimlerin Görselleştirilmesi

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

İmmünkompetan Farelerin Beyinde Non-invaziv Transnazal Yolla İnsan Kaynaklı Pluripotent Kök Hücre Kaynaklı Mikroglia Transplantasyonu

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...