وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح R15 GM061539 2-02 (لوبا)، 2 R15 NS40425-03 (لJEL)، MH 66179 (الدكتور غاري بانكر أوريغون للصحة والعلوم / OHSU)، وP30 NS061800 (للدكتور سو Aicher من OHSU). نشكر باربرا Smoody للحصول على دعم واسع النطاق مع الثقافة من الخلايا العصبية قرن آمون، والدكاترة. بريان لونج وجيمس بيلي لقراءة نقدية لهذه المخطوطة.

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والكتاب ليس لديهم ما يكشف.

ظهرت النخيل وتقنيات الفحص المجهري مضان فائقة الدقة ذات الصلة مؤخرا باعتباره مكملا قيما لأشكال أفضل من تأسيس المجهر الضوئي والمجهر الإلكتروني (EM) 22-24. وتشمل سمات إيجابية من النخيل إعداد نموذج بسيط نسبيا والحد الأدنى من العينة الاضطراب. من حيث المبدأ، PALM يمكن أن ?...

وهناك عدد من العمليات الخلوية تعتمد على دقة وكفاءة الاتجار حويصلة بوساطة الجزيئات الحيوية إلى وجهات التحت خلوية معينة. أحد الأمثلة البارزة هو التجمع متشابك، والذي يسبقه تراوحت فترة طويلة، بوساطة حويصلة متشابك تسليم المكونة من مواقع نشوء حيوي في سوما العصبية إلى مواقع ما قبل وبعد المشبكي يحتمل القاصي 1. مضان المجهري هو وسيلة قوية وشعبية لدراسة الاتجار حويصلة. وتشمل نقاط القوة في أسلوب حساسيتها، والنوعية، والتوافق مع التصوير الحي 2. للأسف، حتى وقت قريب نسبيا، فقد عانت هذه التقنية من ضعف رئيسي واحد، القرار محدودة حيود 2، مما يعوق الدراسات الهياكل ذات أبعاد أصغر من ~ 250 نانومتر. مؤخرا، القرار الوحشي في مضان المجهري تجاوزت حاجز الحيود مع الأخذ ميل مضان فائقة القرارتقنيات croscopy، مثل النخيل 3. القرار الوحشي من النخيل، وعشرات من نانومتر، يعتبر مثاليا لدراسة الحويصلات، والتي لها أبعاد التي تتراوح عادة من 50-250 نانومتر ~ 4. بالتالي فمن الممكن الآن لاستخدام مضان، مع قوتها لا تعد ولا تحصى، لتوضيح مجموعة من الصفات التي يتعذر الوصول إليها سابقا من الحويصلات، بما في ذلك بعض جوانب الاتجار بهم إلى مواقع التحت خلوية معينة. النخيل ليست تافهة لتنفيذ، والاستراتيجيات الناجحة في كثير من الأحيان يجب أن يكون متلائما مع نوع النظام تحت الدراسة. نحن هنا تصف كيفية تنفيذ الدراسات PALM هياكل الحويصلي، وعلينا أن نبرهن على فعالية نهجنا لحالة DCVs في الخلايا العصبية قرن آمون. على وجه الخصوص، ونحن نستخدم النخيل لمعالجة الفرضية القائلة بأن الاتجار DCVs لنقاط الاشتباك العصبي في الخلايا العصبية قرن آمون بوساطة مجموعات DCV 5-8. الاتجار بوساطة مجموعة من الحويصلات إلى نقاط الاشتباك العصبي في تطوير نeurons هو إمكانية مثيرة للاهتمام لأنها قد تسهل تحقيق الاستقرار متشابك السريع والتجمع 9،10. أنصار الاتجار عنقودية من DCVs الاستشهاد حجم واضحا كبير من نقاط والفلورسنت extrasynaptic إيواء البضائع DCV الخارجية كدليل دعم المجموعات 6. ومع ذلك، تظهر هذه الصور في نقاط والتي تم إنشاؤها باستخدام تقنيات المجهر مضان محدودة الحيود، والتي لا تتناسب مع حجم الآثار الناجمة عن حيود المميزة من تلك التي تنشأ من المجموعات. لحل هذه المشكلة، جمعنا مضان widefield التقليدية والصور النخيل من الخلايا العصبية الحصين معربا عن الوهم تستهدف DCVs. وكشف تحليل هذه الصور التي&gt; 92٪ من التجمعات المفترضة DCV extrasynaptic في الصور التقليدية يتم حلها إلى 80 نانومتر (الفردية DCV الحجم) 11 نقاط والنخيل في الصور. وبالتالي، فإن هذه البيانات يبطل إلى حد كبير فرضية التكتل كما ينطبق على DCVs في تطوير hippocaالخلايا العصبية mpal.

<table border="0" cellpadding="0" cellspacing="0" width="1105">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Zeiss PALM</td>
			<td style="width:169px;">Carl Zeiss, Inc.</td>
			<td style="width:119px;">Elyra PS.1</td>
			<td style="width:572px;">With Zeiss Efficient Navigation (ZEN) software and fluorescence filter Set 77 HE GFP/mRFP/Alexa633&#160;</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:245px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:169px;">Life Technoligies</td>
			<td style="width:119px;">11668-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Minimum Essential Medium</td>
			<td style="width:169px;">Life Technologies</td>
			<td style="width:119px;">11095-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Phosphate-Buffered Saline</td>
			<td style="width:169px;">Life Technologies</td>
			<td style="width:119px;">10010049</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Paraformaldehyde</td>
			<td style="width:169px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">19208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sucrose</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:119px;">S-8501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">growth glass coverslips 18mm 1.5D</td>
			<td style="width:169px;">Fisher Scientific</td>
			<td style="width:119px;">NC0059095</td>
			<td></td>
		</tr>
	</tbody>
</table>

super-resolution imaging of neuronal dense-core vesicles

<html> الكشف عن مضان يوفر الأساس لكثير من التقنيات المستخدمة على نطاق واسع المجهري وتتقدم بسرعة المستخدمة في التطبيقات البيولوجية والطبية الحديثة. وتشمل نقاط القوة في مضان حساسيتها، والنوعية، والتوافق مع التصوير الحي. للأسف، الأشكال التقليدية للمضان المجهري يعانون من الضعف، محدودة حيود قرار رئيسي واحد في الطائرة والتصوير، مما يعوق الدراسات الهياكل ذات أبعاد أصغر من ~ 250 نانومتر. مؤخرا، تم التغلب على هذا القيد مع إدخال تقنيات مضان المجهري فائقة القرار، مثل المجهر photoactivated التعريب (النخيل). على عكس نظيراتها التقليدية، ويمكن PALM تنتج صورا مع قرار الوحشي للعشرات من نانومتر. بالتالي فمن الممكن الآن لاستخدام مضان، مع قوتها لا تعد ولا تحصى، لتوضيح مجموعة من الصفات التي يتعذر الوصول إليها سابقا من الهيكل الخلوي والتنظيم. _content &quot;&gt; وللأسف، PALM ليست تافهة لتنفيذ، والاستراتيجيات الناجحة في كثير من الأحيان يجب أن يكون متلائما مع نوع النظام تحت الدراسة. في هذه المقالة، وتبين لنا كيفية تنفيذ الدراسات PALM أحادية اللون البنى حويصلي في الخلايا العصبية مثقف ثابت. يعتبر مثاليا النخيل لدراسة الحويصلات، والتي لها أبعاد التي تتراوح عادة من 50-250 نانومتر ~. الخطوات الرئيسية في نهجنا وصفها تشمل الخلايا العصبية مع photoconvertible (الأخضر إلى الأحمر) الوهم من البضائع حويصلة، وجمع الصور الخام عينات قليلة مع نظام المجهري فائقة القرار، ومعالجة الصور الخام لإنتاج صورة عالية الدقة النخيل، ونحن أيضا تدليل على فعالية نهجنا من خلال تقديم صور استثنائية تحل بشكل جيد من الحويصلات كثيفة الأساسية (DCVs) في الخلايا العصبية الحصين مثقف، الذي دحض الفرضية القائلة بأن الاتجار extrasynaptic من DCVs يتم بوساطة حد كبير من قبل مجموعات DCV.

ويبين الشكل 1 المنتج النهائي واحد من التصوير والتجهيز. في هذه الصورة النخيل، وتظهر الإحداثيات الوحشي fluorophores المترجمة باستخدام وضع العرض النقطه الوسطى، ويظهر فائقة الدقة صورة DCV المرتبطة باستخدام وضع العرض الضبابي. ويبي...

Watch this Scientific Journal Video about Super-resolution Imaging of Neuronal Dense-core Vesicles at JoVE.com

Super-resolution Imaging of Neuronal Dense-core Vesicles

نحن تصف كيفية تنفيذ photoactivated توطين المجهري (النخيل) القائم على الدراسات من الحويصلات في الخلايا العصبية مثقف الثابتة. المكونات الرئيسية لبروتوكول لدينا تشمل وضع العلامات الحويصلات مع الوهم photoconvertible، وجمع الصور الخام عينات قليلة مع نظام المجهر فائقة القرار، ومعالجة الصور الخام لإنتاج فائقة دقة وضوح الصورة.

فائقة الدقة التصوير من الخلايا العصبية الحويصلات كثيفة الأساسية

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

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Neuroscience

9.6K Views.  Lewis & Clark College.  نحن تصف كيفية تنفيذ photoactivated توطين المجهري (النخيل) القائم على الدراسات من الحويصلات في الخلايا العصبية مثقف الثابتة. المكونات الرئيسية لبروتوكول لدينا تشمل وضع العلامات الحويصلات مع الوهم photoconvertible، وجمع الصور الخام عينات قليلة مع نظام ال...

Video: Super-resolution Imaging of Neuronal Dense-core Vesicles

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

الكالسيوم في الجسم الحي التصوير الخلايا العصبية في C. ايليجانس

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

في فيفو التصوير من داور محددة العصبية في إعادة عرض C. ايليجانس

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

الحضانة لفترات طويلة الحادة العصبية الأنسجة للالكهربية والكالسيوم التصوير

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

تحليل الجذعية الصرف في الأعمدة والطبقات

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

تصوير الكالسيوم اثنين-فوتون في Dendrites العصبية في الدماغ شرائح

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

النهج الكمية لتسجيل في الجسم العصبي الكلي الجسمية وبثق Organelle في Vesicles كبيرة exopher في C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

التصوير فائق الدقة لدراسة التعريب المشترك للبروتينات وعلامات متشابك في الخلايا العصبية الأولية

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

إعداد الكبار Drosophila melanogaster لتصوير الدماغ كله أثناء السلوك والمحفزات الاستجابات

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

ROS تصوير الخلايا الحية أثناء تطوير الخلايا العصبية

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

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.