ويؤيد الأمين العام بأموال من جامعة ولاية نيويورك في بوفالو من مؤسسة جون وOishei ر. وأيد عضو الكنيست من قبل الطلاب جائزة البحوث التي CURCA في UB.

1. إعداد الكواشف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد إعداد العازلة تشريح بإضافة مركبات التالية : 128 مم كلوريد الصوديوم ، 1MM EGTA ، 4 مم MgCl 2 ، 2mm وبوكل ، HEPES 5mm ، و السكروز و 36 ملم في 1000 PH ، إلى 7.2 مل وتصفية تعقيم. </li></ol> 2. التحضير للتشريح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Siligard المكعب : هو قطع الجل إلى حوالي بوصة واحدة واسعة ، 1 بوصة طويلة وحوالي 1cm عالية. يتم وضع المكعب الجل على شريحة زجاجية أثناء تشريح. ويمكن استخدام مكعبات أكثر من مرة. </li><li style=";text-align:right;direction:rtl"> دبابيس : دبابيس تستخدم لتشريح يلزم اضافية قصيرة. في الجزء السفلي من المسامير الحادة minutien يعمل بشكل أفضل في تشريح. </li><li style=";text-align:right;direction:rtl"> حاجة واحدة في الربيع microdissection حادة المقص. </li><li style=";text-align:right;direction:rtl"> تحتاج اثنين ملقط يميل بشكل جيد. </li></ol> 3. جمع اليرقات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 3 تجول اليرقات طور مرحلي الثالثة التي تعبر عن البروتين GFP حويصلة الموسومة يتم جمعها على طبق بتري. </li><li style=";text-align:right;direction:rtl"> تغسل في ماء منزوع الأيونات اليرقات للتخلص من جميع المواد الغذائية. </li></ol> 4. تعيش تشريح اليرقات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم نقل يرقة إلى تشريح مكعب الجل على شريحة زجاجية. غير منغمسين في اليرقة العازلة تشريح. </li><li style=";text-align:right;direction:rtl"> وموهون يرقة في الأمامي والخلفي باستخدام اثنين من الدبابيس بشكل جيد مع الجانب الظهري تصل. </li><li style=";text-align:right;direction:rtl"> باستخدام مقص microdissection ، يتم إجراء قطع صغير في المنتصف قرب نهاية الخلفي. من هذا يتم إجراء شق طريق إلى قطع الأمامية. باستخدام اثنين من دبابيس ، دبوس إهاب قطع على الحواف الجانبية. </li><li style=";text-align:right;direction:rtl"> إضافة قطرة من تشريح العازلة. إزالة بعناية القناة الهضمية والأمعاء والهيئات الدهون بالملقط. معظم هذه الأنسجة طين عادة من خلال القطع الظهرية. </li><li style=";text-align:right;direction:rtl"> استبدال العازلة مع الطازجة العازلة تشريح. يجب أن تكون جافة اليرقة أبدا ، وينبغي أن يكون دائما في تشريح العازلة. </li><li style=";text-align:right;direction:rtl"> تتم حضانات تستخدم في الجسم الحي أو علامات MitoTracker LysoTracker في هذا الوقت ، من خلال تمييع في علامة حية في تشريح العازلة. بعد حضانة في علامة فيفو غسل اليرقة تشريح عدة مرات (5 يغسل سريع) باستخدام الطازجة العازلة تشريح. </li><li style=";text-align:right;direction:rtl"> بعد استبدال غسل الماضي دفع المسامير في syligard ساترة وعلى الفور. اليرقات جاهز الآن للمراقبة. </li></ol> 5. تعيش اليرقات التصوير تشريح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضع ساترة والجل على خشبة المسرح والصورة باستخدام مجهر مجهر مقلوب. </li><li style=";text-align:right;direction:rtl"> نستخدم عدسة 100X إلى الحويصلات صورة الموسومة الأعصاب داخل القطعة اليرقات. هو أولا تقييم GFP التعبير في الهيئات خلية من خلايا العقدة البطنية قبل أن يتم تصوير الأعصاب قطعي. إذا لوحظ قوية تلطيخ GFP في الهيئات خلية في بطني العقدة ، ثم يتم تصوير الأعصاب واستخدامها في التقييم. </li><li style=";text-align:right;direction:rtl"> باستخدام نظام عرض ثنائي مع المرشحات لCFP / YFP أو YFP RFP / وتقسيم البرنامج عرض في وقت واحد يمكننا صورة اثنين من البروتين المفتاحية في الجسم الحي. </li></ol> 6. ممثل النتائج <img src="/files/ftp_upload/2151/2151fig1.jpg" alt="الشكل 1" /> الشكل 1 : صورة تظهر حويصلات ممثل محواري محواري كثيرة على مسارات عديدة في عصب قطعي اليرقات لاحظ النقط GFP ، تمثل تراكمات محواري. <img src="/files/ftp_upload/2151/2151fig2.jpg" alt="الشكل 2" /> الشكل 2 : صورة ممثل يظهر مسار واحد مع محواري محواري حويصلات عديدة داخل عصب قطعي اليرقات.

<ol>
	<li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Hurd, D. D., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+mutations+cause+motor+neuron+disease+phenotypes+by+disrupting+fast+axonal+transport+in+Drosophila.">Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila.</a> Genetics. 144, 1075-1085 (1996).</li><li>Gunawardena, S., Her, L. S., Brusch, R. G., Laymon, R. A., Niesman, I. R., Gordesky-Gold, B., Sintasath, L., Bonini, N. M., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+axonal+transport+by+loss+of+huntingtin+or+expression+of+pathogenic+polyQ+proteins+in+Drosophila.">Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila.</a> Neuron. 40, 25-40 (2003).</li></ol>

الإعلان عن أي تضارب في المصالح.

في مجال التصوير فيفو النقل حويصلة متشابك داخل الأعصاب يرقات ذبابة الفاكهة قطعي هو أداة قوية لدراسة آليات النقل في محواري. لقد اعتدنا في السابق على هذا البروتوكول لتقييم ديناميات النقل داخل الأعصاب اليرقات يكرر الإعراب عن توسيع polyQ لتوضيح كيفية توسيع ي...

<table><tbody><tr><td>1 Pair Micro Dissection Scissors</td><td>Fine Science Tools</td><td></td><td>Spring scissors &amp;#8211; 6mm blade. 0.125 mm tip diameter</td></tr><tr><td>2 Pair Forceps</td><td>Fisher Scientific</td><td></td><td>Fisherbrand Dissecting Micro-Adson serrated tip forceps</td></tr><tr><td>Box of Dissection Pins</td><td>Fine Science Tools</td><td></td><td>Minutien Pins 0.1mm diameter</td></tr></tbody></table>

in vivo visualization of synaptic vesicles within drosophila larval segmental axons

توضيح آليات النقل محواري أثبتت أنها مهمة جدا في تحديد كيفية العيوب في وسائل النقل لمسافات طويلة تؤثر أمراض عصبية مختلفة. يمكن عيوب أساسية في هذه العملية لها آثار ضارة على أداء الخلايا العصبية والتنمية. قمنا بتطوير بروتوكول التشريح الذي تم تصميمه لفضح<em&gt; ذبابة الفاكهة</em&gt; الأعصاب قطعي اليرقات لعرض المحاور النقل في الوقت الحقيقي. وقد تكيفت على هذا البروتوكول لأننا نعيش من تصوير واحد نشرته هيرد وساكستون (1996) تستخدم لimmunolocalizatin الأعصاب قطعي اليرقات. تشريح دقيق والظروف المناسبة العازلة ذات أهمية حاسمة لتحقيق أقصى قدر من عمر اليرقات تشريح. عندما يوظف بشكل صحيح ، وقد أظهرت يرقات تشريح قوية حويصلة النقل لمدة 2-3 ساعات في ظل الظروف الفسيولوجية. نستخدم أسلوب UAS - GAL4<sup&gt; 1</sup&gt; للتعبير عن GFP الموسومة APP أو حويصلات synaptotagmin ضمن محور عصبي واحد أو العديد من المحاور في الأعصاب قطعي اليرقات باستخدام مختلف الخلايا العصبية GAL4 السائقين. علامات أخرى الموسومة fluorescently ، على سبيل المثال mitochrondria (MitoTracker) أو lysosomes (LysoTracker) ، يمكن أن ينطبق أيضا على اليرقات قبل المشاهدين. ويمكن الاطلاع GFP - حويصلة الحركة وحركة الجسيمات في وقت واحد باستخدام موجات منفصلة.

Watch this Scientific Journal Video about In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons at JoVE.com

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

يناقش هذا البروتوكول تشريح حية<em&gt; ذبابة الفاكهة</em&gt; اليرقات لغرض التصوير حركة الحويصلات GFP محواري الموسومة على المسارات أنيبيب.

في التصور المجراة من الحويصلات متشابك ضمن محاور عصبية ذبابة الفاكهة القطاعي اليرقات

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

in-vivo-visualization-synaptic-vesicles-within-drosophila-larval

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

9.4K Views.  SUNY-University at Buffalo. يناقش هذا البروتوكول تشريح حية ذبابة الفاكهة اليرقات لغرض التصوير حركة الحويصلات GFP محواري الموسومة على المسارات أنيبيب.

Video: In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

تصور الأعصاب القطاعي اليرقات في 3 الثالثة طور مرحلي ذبابة الفاكهة التحضيرات اليرقات

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

تشريح والتصوير من المناطق النشطة في ذبابة الفاكهة الوصل العصبي العضلي

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

باستخدام رقائق الموائع الدقيقة للتصوير لايف ودراسة الردود الإصابات في<em&gt; ذبابة الفاكهة</em&gt; يرقات

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Bioengineering

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

تصور لايف ذبابة الفاكهة الدبقية العصبية والعضلية ، تقاطع مع الأصباغ نيون

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Biology

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

تصور المورفولوجية الساق الحركية محاور عصبية من خلال بشرة الكبار

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Developmental Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

في التصوير من يرقات ذبابة الفاكهة فيفو سليمة الفرعية الخلوية في القرار

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

التصور من محور الإسقاط نمط من الخلايا العصبية المحرك الجنينية في<em&gt; ذبابة الفاكهة</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

في فيفو تتبع في محطة Presynaptic الأعصاب الحركية المورفولوجية جزيء واحد

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

الفاصل الزمني التصوير الحي والقياس الكمي لديناميات فرع التقشر السريع في تطوير الخلايا العصبية Drosophila

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

في التصور المجراة من الحويصلات متشابك ضمن محاور عصبية ذبابة الفاكهة القطاعي اليرقات

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تصور الأعصاب القطاعي اليرقات في 3^{الثالثة} طور مرحلي ذبابة الفاكهة التحضيرات اليرقات