We are grateful to Dr. Sachiko Sato and Julie-Christine L&#233;vesque at the Bioimaging Platform of the Centre de recherche du CHU de Qu&#233;bec for their technical assistance. Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2014-05308, The Banting Research Foundation, and The Scottish Rite Charitable Foundation of Canada to M.E.T supported this work.
H.E.H. is recipient of a scholarship from the Lebanese Ministry of Education and Higher Education, and K.B. from the Facult&#233; de m&#233;decine of Universit&#233; Laval.

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The authors have nothing to disclose.

Этот протокол объясняет корреляционный подход к ориентации плотного ядра A &amp; beta; бляхи с ЕМ. Метокси-X04 в естественных условиях инъекции позволяет быстро выбор участков мозга , которые содержат A &amp; beta ; бляшками в пределах отдельных регионов и слоев , представляющих интерес, напри...

Амилоида A &amp; beta; формирование бляшек является основным нейропатологических признаком болезни Альцгеймера (AD). Тем не менее все больше доказательств свидетельствует о важной роли иммунной системы в прогрессировании заболевания у детей в 1,2 раза . В частности, новые данные из доклинических и клинических исследований установлено, дисфункции иммунной системы в качестве основного водителя и вкладчика в AD патологии. С этими выводами, центральной и периферической иммунные клетки появились в качестве перспективных терапевтических мишеней для 3 AD. Следующий протокол сочетает в себе световой и электронной микроскопии (ЭМ) для создания новое понимание взаимосвязи между Ар осаждения бляшек и микроглии фенотипических изменений в AD. Этот протокол позволяет маркировать A &amp; beta ; бляшек в мышиных моделях AD с использованием в естественных условиях инъекции флуоресцентного красителя метокси-X04. Метокси-X04 является конго красным производным инструментом, который может легко пересечь гематоэнцефалический барьер, чтобы войти в паренхиму мозга и связывать β-складчатой ​​листы с высокой аffinity. Поскольку краситель флуоресцентный, он может быть использован для обнаружения в естественных условиях осаждения бляшек Ар с двухфотонной микроскопии 4. После того, как связан с A &amp; beta;, метокси-X04 не диссоциирует или перераспределить от бляшек, и он сохраняет свою флуоресценцию с течением времени. Это , как правило , вводят периферически , чтобы позволить неинвазивной визуализации динамики мозга 5. Флуоресценции также остается следующая альдегидной фиксации, что позволяет анализирует соотносительны некропсический, включая расследование гибели нейронов в районе Ар бляшек 6. Этот протокол использует свойств метокси-X04 , чтобы выбрать разделы мозга от APP SWE / PS1A 246E мышей (APP-PS1; совместной экспрессии двойной мутации в APP гене Lys670Asn / Met671Leu и вариант пресенилин PS1-A264E человек) 7 , которые проявляют A &amp; beta бляшки в конкретных регионах, представляющих интерес (гиппокампа СА1, пластового radiatum и lacunosum-moleculare) До предварительно встраивания иммуноокрашивания против микроглии маркера ионизированного кальция связывающей молекулы адаптер 1 (Iba1) для визуализации микроглии органы и процессы с EM клеток. Мышам дают внутрибрюшинную инъекцию раствора метокси-x04, 24 ч до начала фиксации мозга через transcardial перфузией. Коронарные срезы мозга получены с использованием vibratome. Разделы, содержащие гиппокампа CA1 подвергают скринингу под флуоресцентным микроскопом на наличие бляшек A &amp; beta; в слоях radiatum и lacunosum-moleculare. Иммуногистохимическое Iba1, четырехокись осмия после фиксации и пластиковой смолы вложение затем выполняются на отдельных участках мозга. В конце этого протокола, секции могут быть сохранены без дальнейшей деградации ультраструктурным, готовый к ультратонкой секционирования и ультраструктурным экспертизы. Важно отметить, что бл шки еще флуоресцентный после иммунным с различными антителами, например Iba1, как в настоящем протоколе. Они становятся темнее, тхань окружающих их нейропиля следующие четырехокись осмия после фиксации, независимо от метокси-X04 окрашивания, что позволяет точно определить регионы, представляющие интерес, как правило, до нескольких квадратных миллиметров, чтобы быть исследованы с помощью просвечивающего электронного микроскопа. Этот соотносительны подход предлагает эффективный способ идентификации конкретных участков мозга для изучения на ультраструктурном уровне. Это особенно полезно при изучении ранней патологии AD, в пределах определенных областей головного мозга или слоев, которые могут содержать только несколько A &amp; beta; бляшками, присутствует только небольшая часть срезов ткани. В это время особенно, было бы неэффективно использовать иммуногистохимическое (A &amp; beta; и двойной маркировки для других клеточных маркеров, таких как Iba1) на нескольких срезах мозга просто, чтобы получить небольшую фракцию, содержащую A &amp; beta; бляшками в нужном месте. Кроме того, инъекции живых мышей с метокси-X04 перед умерщвлением и обработки тканей не Специальное соглашениеЕ ультраструктурную сохранение. Альтернативные методы , такие как посмертного окрашивания Конго красным Тиофлавин S, Тиофлавин T или метокси-X04 на участках фиксированных тканей требуют окрашивания дифференциации в этаноле, 8-11 , который вызывает осмотический стресс и разрушает ультраструктуры. Конго красный также является известным канцерогеном для человека 12.

<table border="0" cellpadding="0" cellspacing="0" width="1006">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Methoxy-X04</td>
			<td style="width:197px;">Tocris Bioscience</td>
			<td style="width:119px;">4920</td>
			<td style="width:167px;">10 mg substrate per tablet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene glycol</td>
			<td>Sigma Aldrich</td>
			<td>W294004&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher BioReagents</td>
			<td>BP231-1</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP153500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrolein&#160;</td>
			<td>Sigma</td>
			<td>110221</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde Granular</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper</td>
			<td>Fisher</td>
			<td>09-790-14F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump with Tubing</td>
			<td>ColeParmer</td>
			<td>cp.78023-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel Winged blood Collection Set Needles 25G&#160;</td>
			<td>Becton Dickinson</td>
			<td>367341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extrafine Forceps</td>
			<td>F.S.T</td>
			<td>11152-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td>F.S.T</td>
			<td>13003-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors</td>
			<td>F.S.T</td>
			<td>14004-16</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Dissecting Scissors</td>
			<td>ROBOZ surgical store</td>
			<td>5818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>74515-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibrating Blade Michrotome Leica VT1000 S</td>
			<td>Leica Biosystems&#160;</td>
			<td>14047235612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome blades</td>
			<td>Electron microscopy Sciences</td>
			<td>71990</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene Glycol</td>
			<td>Fisher BioReagents</td>
			<td>BP230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen Peroxide, 30%</td>
			<td>J.T.BAKER</td>
			<td>cat: 2186-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium borohydride</td>
			<td>Sigma</td>
			<td>480886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl</td>
			<td>Fisher</td>
			<td>BP153-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine Serum (FBS)</td>
			<td>Sigma Aldrich</td>
			<td>F1051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA), fraction V</td>
			<td>Thomas Scientific</td>
			<td>C001H24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti IBA1, Rabbit&#160;</td>
			<td>WAKO</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>111066046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASTAIN Elite ABC Kit (Standard)</td>
			<td>Vector Labs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.3&#39;-Diaminobenzidine tetra-hydrochloride (DAB)</td>
			<td>Sigma</td>
			<td>D5905-50TAB</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osmium tetroxide, 4% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>19150</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component A</td>
			<td>Sigma</td>
			<td>44611</td>
			<td>Resin Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component B</td>
			<td>Sigma</td>
			<td>44612</td>
			<td>Hardener Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component C</td>
			<td>Sigma</td>
			<td>44613</td>
			<td>Plasticizer Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component D</td>
			<td>Sigma</td>
			<td>44614</td>
			<td>Accelerator Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>LesAlcoolsdeComerce</td>
			<td>151-01-15N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene oxide</td>
			<td>Sigma</td>
			<td>110205</td>
			<td>Caution: corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum weigh dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>70048-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACLAR&#174;&#8211;Fluoropolymer Films</td>
			<td>Electron Microscopy Sciences</td>
			<td>50425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oven/Incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques

Подробный протокол приводится здесь для выявления амилоида A &amp; beta; бляшек в срезах мозга от мышиных моделей болезни Альцгеймера до того перед вложением иммуноокрашивания (специально для ионизированного кальция связывания адаптера молекулы 1 (Iba1), кальций-связывающий белок выражается микроглии) и обработки тканей для электронная микроскопия (ЭМ). Метокси-X04 представляет собой флуоресцентный краситель, который пересекает гематоэнцефалический барьер и избирательно связывается с бета-плиссе листов найдены в плотном ядре A &amp; beta бляшек. Инъекция животных с метокси-X04 до умерщвления и фиксации мозга позволяет предварительный отбор и отбор бляшек, содержащих срезов мозга для дальнейшей обработки с трудоемких манипуляций. Это особенно полезно при изучении ранней патологии AD в пределах определенных областей головного мозга или слоев, которые могут содержать очень мало бляшек, присутствующих в лишь небольшая часть из секций. обработка Посмертное срезов ткани Конго красным Тиофлавин S и ThioflAvin T (или даже с метокси-x04) может пометить бета-складчатой ​​листов, но требует обширных клиринг с этанолом для удаления избытка красителя и эти процедуры несовместимы с сохранением ультраструктурным. Было бы также неэффективным для выполнения маркировки на A &amp; beta; (и других клеточных маркеров, таких как Iba1) на всех участках мозга из регионов, представляющих интерес, только, чтобы получить небольшую фракцию, содержащую A &amp; beta; бляшками в нужном месте. Важно отметить, что Ар бляшек все еще видны после обработки ткани для ЭМ, что позволяет для точной идентификации областей (как правило, до нескольких квадратных миллиметров) для изучения с помощью электронного микроскопа.

В данном разделе представлены результаты, которые могут быть получены при различных критических шагов протокола. В частности, результаты показывают примеры срезов мозга, содержащих метокси-X04 окрашивали бляшек в конкретном регионе и слои, представляющие интерес: гип...

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃŽÂ²-Amyloid Plaques at JoVE.com

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

В данной статье описывается протокол для визуализации амилоидных бляшек A &amp; beta; в мышиных моделях болезни Альцгеймера с помощью метокси-X04, который пересекает гематоэнцефалический барьер и избирательно связывается с бета-складчатые листы, найденные в плотном ядре A &amp; beta бляшек. Это позволяет предварительный отбор срезов ткани зубной налет, содержащий до иммунной окраски и обработки для электронной микроскопии.

Коррелятивное световой и электронной микроскопии для изучения микроглии взаимодействий с бета-амилоидных бляшек

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

correlative-light-electron-microscopy-to-study-microglial

Research

JoVE Journal

Neuroscience

11.2K Views.  CHU de Québec Research Center.  В данной статье описывается протокол для визуализации амилоидных бляшек A & beta; в мышиных моделях болезни Альцгеймера с помощью метокси-X04, который пересекает гематоэнцефалический барьер и избирательно связывается с бета-складчатые листы, найденные в плот?...

Video: Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

Сосредоточены ионного пучка фрезерные и сканирующей электронной микроскопии ткани мозга

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Свет Анализ предпочтений по изучению врожденных и суточного Регулируемые Photobehavior в<em&gt; Drosophila</em&gt; Личинки

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Микронного масштаба Разрешение оптической томографии целых мозг мышей с конфокальной Light Sheet микроскопии

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Генетических манипуляций мозжечка Granule нейронов<em&gt; Экстракорпоральное</em&gt; И<em&gt; В Vivo</em&gt; По изучению нейронных Морфология и миграция

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Обонятельная система как модель для изучения характера роста аксонов и морфология<em&gt; В Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

Мозг человека кровь интерфейс модель для изучения пересечения барьера патогенов или лекарства и их взаимодействия с мозгом

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Светиндуцированная молекулярная адсорбция белков с использованием системы PRIMO для микро-паттернинга для изучения реакции клеток на внеклеточные матричные белки

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

Последовательная блочно-лицевая сканирующая электронная микроскопия (SBEM) для исследования дендритных шипов

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

Первичные культуры астроцитов и микроглии крыс и их использование в изучении бокового амиотрофического склероза

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Когерентная антистоковская рамановская спектроскопия (CARS) для визуализации миелинизации в срезах мозга

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

Коррелятивное световой и электронной микроскопии для изучения микроглии взаимодействий с бета-амилоидных бляшек

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