Name: acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective
Uploaded: 2014-10-04T15:00:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective at JoVE.com

Gli autori desiderano ringraziare Alexandra Opie per l&#39;assistenza e il supporto tecnico, James Barton per la fornitura di video narrazione, e Ramona Flatz e Joellen Sweeney per apparire nel video allegato. Questo lavoro è stato supportato dal Charitable Trust MJ Murdock (Grant No. 2.012.205 a JED).

1 Il sistema Microscopio Stereo Nota: Anche se questo protocollo è facilmente adattabile a qualsiasi impianto stereo microscopio accoppiato ad una fotocamera digitale con la possibilità di acquisire video, dettagli sono riportati sul sistema utilizzato nel nostro laboratorio (Tabella dei materiali / attrezzature). <ol><li> Acquisire video digitale utilizzando uno stereo microscopio trinoculare accoppiato ad una fotocamera digitale commerciale. </li><li> Per accoppiare la fotocamera digitale commerciale alla porta trinoculare del microscopio stereo, rimuovere il ½x C-mount del porto fototubo del microscopio stereo e sostituirlo con un 1X C-mount. </li><li> Montare un accoppiatore fotocamera digitale (filetto 43 millimetri) per la 1X C-mount. </li><li> Montare due anelli step-down, 58 mm a 48 mm, e 48 mm a 43 mm, per l&#39;accoppiatore fotocamera per colmare la connessione dal accoppiatore fotocamera digitale ad un kit adattatore per lenti per la fotocamera digitale. </li><li> Montare la fotocamera digitale al kit adattatore per lenti. </li><li> Acquisire video con l&#39;ingrandimento del microscopio e lo zoom ottico della fotocamera digitale impostata per un ingrandimento totale di circa 12X (30 fotogrammi al secondo, 640 x 480 pixel). Nota: L&#39;ingrandimento del microscopio stereo deve essere compensato secondo la nuova riconfigurato 1X C-mount della porta trinoculare. </li></ol> 2 Imaging Drosophila larve terzo instar <ol><li> Nastro un pennarello indelebile al piattello nero di un microscopio stereo accoppiato ad una fotocamera digitale in modo che il lato del tappo marcatore occupa circa ⅓ a ¼ del campo di vista verticale osservata nel monitor LCD della fotocamera. Utilizzare cime marcatori come il palco per eseguire l&#39;imaging larvale perché vengono in un assortimento di colori che può essere utilizzato per codice colore e differenziare i genotipi di larve di essere ripreso. </li><li> Delimitare il campo di vista osservato nel monitor LCD della fotocamera digitale sulla superficie del piano marcatore con una punta finemarcatore. </li><li> Selezionare un terzo stadio larva di immagine. I criteri per la selezione larve al terzo stadio era lunghezza del corpo, emergere dalla fonte di cibo durante la fase larvale del ciclo di vita, la presenza di spiracoli anteriori e posteriori, e la struttura dei ganci mandibolari dell&#39;apparato bocca 11. Assicurarsi che la larva è pulito lavandolo accuratamente con acqua. </li><li> Illuminare la fase superiore pennarello indelebile dall&#39;alto con luce da un sistema di illuminazione a fibre ottiche. Regolare l&#39;angolazione della luce incidente per fornire l&#39;illuminazione ottimale. </li><li> Mettere a fuoco il microscopio sul bordo del piano pennarello indelebile. Iniziare l&#39;acquisizione video digitale. </li><li> Posizionare la larva sul lato del tappo marcatore di circa 75 ° rispetto all&#39;asse verticale, appena fuori dal campo visivo, con l&#39;anteriore della larva rivolta verso il campo di vista (Figura 1). Nota: Posizionamento della larva sul lato del tappo indicatore permette alla fotocamera di registrare il movimento di the larva da una prospettiva laterale. Aiuta a mantenere la larva umido con acqua in modo che non cadano fuori il lato del tappo marcatore. Prestare attenzione, tuttavia, non usare troppa acqua come quantità eccessive potranno aderire alla larva come si esegue la scansione attraverso il campo. </li><li> Colpire delicatamente e spingere la larva con un piccolo pennello per costringere a strisciare attraverso il campo di vista. Sii paziente come le larve raramente cooperano e spesso devono essere restituiti al punto di partenza molte volte prima che strisciano attraverso il campo. </li><li> Record di circa 10-15 minuti di ininterrotta riprese video digitali e ritagliare e rimuovere tutti i filmati inutili post-acquisizione digitale con software di editing video. </li></ol> 3. Imaging adulti Drosophila <ol><li> Posizionare un singolo adulto Drosophila in un usa e getta da 1,5 ml spettroscopica polistirolo cuvetta. Nota: CO 2 per l&#39;anestesia di adulti Drosophila immediatamente prima di un Behavprotocollo di analisi ioral può compromettere i risultati 12. Si raccomanda di adulti Drosophila disporre di un periodo di 24 ore per recuperare da CO 2 l&#39;anestesia prima di eseguire un test comportamentale 13. </li><li> Inserire l&#39;estremità della cuvetta con un piccolo batuffolo di cotone. Assicurarsi che il batuffolo di cotone è ricco abbastanza stretto per occupare il grande spazio tappo e limita la mosca al vano ridotto volume della cuvetta. </li><li> Inserire la cuvetta sul piatto bianco fase di un microscopio stereo e allineare correttamente il vano ridotto volume della cuvetta con il campo visivo osservata nel monitor LCD della fotocamera digitale. </li><li> Illuminare la cuvetta dall&#39;alto con luce da un sistema di illuminazione a fibre ottiche. Regolare l&#39;angolazione della luce incidente per fornire l&#39;illuminazione ottimale. </li><li> Mettere a fuoco il microscopio e iniziare l&#39;acquisizione di video digitale. </li><li> Record di circa 30-45 min di ininterrotta riprese video digitali e ritagliare e rimuovere tutto il superfluorepertorio post-acquisizione digitale con software di editing video. </li></ol>

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Forza Drosophila melanogaster s &#39;come sistema modello per lo studio della funzione del sistema nervoso deriva in gran parte dalla convergenza dei potenti strumenti genetici disponibili e la vasta gamma di test comportamentali robusti sviluppato. Qui vi presentiamo una tecnica di microscopia semplice e ampiamente accessibili per l&#39;acquisizione video digitale di alta qualità di Drosophila adulti e fenotipi locomotiva larvali da una prospettiva laterale. Abbiamo utilizzato con successo questo app...

Il frutto comune mosca Drosophila melanogaster è un potente sistema modello sperimentale per lo studio della funzione del sistema nervoso 1-3. Conservazione evolutiva di struttura e funzione del sistema nervoso con gli esseri umani, così come la facilità di manipolazione genetica e di una vasta gamma di strumenti genetici rende Drosophila l&#39;organismo prima di modellare le malattie neurodegenerative umane 4. Mutazioni genetiche che causano la disfunzione del sistema nervoso causano spesso vitale larve mutanti e adulti Drosophila con locomozione compromessa. Fenotipi osservati nel sistema nervoso mutanti difettosi includono il ridotto tasso di locomozione, coordinamento aberrante, e movimenti spastici negli adulti, così come deficit in contrazione peristaltica della muscolatura della parete del corpo, e la paralisi parziale delle larve. Questi fenotipi sono state sfruttate nello sviluppo di high-throughput schermi genetici e test di locomozione di larve mutanti 5, 6 e 7-10 adulto Drosophila volta a quantificare la perdita di valore locomozione e l&#39;identificazione dei geni necessari per il funzionamento del sistema nervoso. Mentre questi approcci sono estremamente utili per quantificare i comportamenti locomotive larvali e adulti, non riescono a trasmettere informazioni qualitative su ogni comportamento problema specifico. Ad esempio, mentre il mutante terzo instar larve può presentare parametri di locomozione alterati in un test comportamentale, può essere chiaro se questo è il risultato di alterazioni contrazioni peristaltiche ritmiche durante il ciclo di scansione, generale mancanza di coordinamento, o paralisi parziale del corpo posteriore muscolatura della parete. Qui si descrive una tecnica di microscopia semplice e ampiamente accessibili per l&#39;acquisizione video digitale di alta qualità di Drosophila adulti e fenotipi locomotiva larvali da una prospettiva laterale. Il video digitale acquisito da una prospettiva laterale, permette l&#39;osservazione diretta e l&#39;analisi delle distinzioni sottili LocomotivE i comportamenti di un più informativo orientamento side-view.

<table><tbody><tr><td>Trinocular Stereozoom Microscope</td><td>Olympus Corporation</td><td>SZ6145TR</td><td>&amp;frac12; C-mount was removed and replaced with 1X C-mount</td></tr><tr><td>1X C-mount</td><td>Leeds Precision Instruments</td><td>LSZ-1XCMT2</td><td></td></tr><tr><td>Digital Camera Coupler (43 mm thread)</td><td>Qioptiq Imaging Solutions</td><td>25-70-10-02</td><td></td></tr><tr><td>58 mm to 48 mm Step Down Ring</td><td>B&amp;amp;H Video</td><td>GBSDR5848</td><td></td></tr><tr><td>48 mm to 43 mm Step Down Ring</td><td>B&amp;amp;H Video</td><td>GBSDR4843</td><td></td></tr><tr><td>Lensmate Adapter Kit for Canon G10</td><td>LensMateOnline.com</td><td></td><td></td></tr><tr><td>Canon PowerShot G10 Digital Camera</td><td>Canon U.S.A., Inc.</td><td></td><td></td></tr><tr><td>1.5 ml Spectroscopic Polysterene Cuvette</td><td>Denville Scientific</td><td>U8650-4</td><td></td></tr></tbody></table>

acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective

Drosophila melanogaster è un potente sistema modello sperimentale per studiare la funzione del sistema nervoso. Mutazioni genetiche che causano la disfunzione del sistema nervoso spesso producono larve e gli adulti che hanno locomozione fenotipi difettosi che sono difficili da descrivere adeguatamente con il testo o completamente rappresentare con una sola immagine fotografica praticabile. Modalità attuali di pubblicazioni scientifiche, tuttavia, sostengono la presentazione di supporti video digitali come materiale supplementare per accompagnare un manoscritto. Qui si descrive una tecnica di microscopia semplice e ampiamente accessibili per l&#39;acquisizione video digitale di alta qualità di entrambi i fenotipi larve e adulti di Drosophila da una prospettiva laterale. Video di locomozione larvale e adulto da una vista laterale è vantaggioso perché permette l&#39;osservazione e l&#39;analisi delle sottili distinzioni e le variazioni nei comportamenti aberranti locomotiva. Abbiamo utilizzato con successo la tecnica di visualizzare e quantificare aberrant strisciare comportamenti terzo instar larve, oltre a fenotipi mutanti adulti e comportamenti, tra cui governare.

Abbiamo utilizzato con successo questa tecnica per acquisire e quantificare il fenotipo larvale comportamentale associata alla perdita di funzione del gene Stathmin (Figura 2) 14. Il gene codifica per una proteina Stathmin normativo microtubuli che le partizioni dimeri di tubulina da pool di tubulina solubile, e si lega i microtubuli e promuove il loro smontaggio 15,16. Funzione Stathmin è necessario per mantenere l&#39;integrità dei microtubuli nelle assoni dei...

Watch this Scientific Journal Video about Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective at JoVE.com

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Qui si descrive una tecnica di microscopia semplice e ampiamente accessibili per acquisire video digitale di alta qualità di Drosophila adulti e fenotipi mutanti larvali da una prospettiva laterale.

Acquisizione di alta qualità video digitale di<em&gt; Drosophila</em&gt; Comportamenti larvali e adulti da una prospettiva laterale

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

acquisition-high-quality-digital-video-drosophila-larval-adult

Research

JoVE Journal

Neuroscience

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

8.7K Views.  Willamette University.  Qui si descrive una tecnica di microscopia semplice e ampiamente accessibili per acquisire video digitale di alta qualità di Drosophila adulti e fenotipi mutanti larvali da una prospettiva laterale. 

Video: Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imaging del<em&gt; Drosophila</em&gt; Pupa Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Ricerca

Biologia dello sviluppo

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Monitoraggio ad alta risoluzione video di locomozione per adulti in Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Biologia

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. &#216;15 x 5mm (no food inside) or &#216;15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of &#8805;30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

Un sistema di imaging ad alta definizione regolabile per studi comportamentali di adulti drosofili

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but ...

Comportamento

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Imaging e analisi dell'orientamento dei tessuti e delle dinamiche di crescita nell'epiteliale della Drosophila in via di sviluppo durante le fasi pupali

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Una semplice tecnica per analizzare l'attività locomotoria nella Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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.

Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

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

<meta charset="utf8"></head><body>Imaging dal vivo esteso della morfogenesi della zampa pupale di Drosophila senza pupario

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

<meta charset="utf8"></head><body>Autore in primo piano: Scoprire le strutture epiteliali dinamiche durante lo sviluppo della Drosophila

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

Imaging dal vivo a lungo termine dello sviluppo della zampa pupale di Drosophila dopo la rimozione del pupario

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

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.

Time-lapse Live Imaging e quantificazione delle dinamiche del ramo dendritico veloce nello sviluppo di neuroni della Drosophila

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

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Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

Imaging dal vivo a lungo termine dello sviluppo della zampa pupale di Drosophila dopo la rimozione del pupario

Imaging dal vivo a lungo termine dello sviluppo della zampa pupale di Drosophila dopo la rimozione del pupario

Imaging dal vivo a lungo termine dello sviluppo della zampa pupale di Drosophila dopo la rimozione del pupario

Time-lapse Live Imaging e quantificazione delle dinamiche del ramo dendritico veloce nello sviluppo di neuroni della Drosophila