Riconosciamo il magazzino centrale di Vienna Drosophila Resource Center e Bloomington Drosophila (NIH P40OD018537) per la fornitura di ceppi di Drosophila. Ringraziamo Jack Fransen dalla Microscopical Imaging Center per il supporto di esperti nel campo dell&#39;imaging. Questo studio è stato sostenuto da VIDI e le sovvenzioni TOP (917-96-346, 912-12-109) dalla Organizzazione olandese per la ricerca scientifica (NWO), da due borse di studio di dottorato di ricerca DCN / Radboud University Medical Center, dal ritardo mentale rete tedesca finanziato dal programma NGFN + del ministero federale tedesco dell&#39;Istruzione e della ricerca (BMBF) e dal Gencodys dell&#39;Unione europea del 7 ° PQ su larga scala integrati di rete (Health-241995) per AS. I finanziatori avevano alcun ruolo nel disegno dello studio, la raccolta e l&#39;analisi dei dati, la decisione di pubblicare, o preparazione del manoscritto.

<ol>
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&quot;Drosophila NMJ Morfometria&quot; e &quot;Drosophila NMJ bouton Morfometria&quot; sono strumenti potenti per i ricercatori interessati a valutare sinapsi morfologia. la valutazione manuale dei parametri NMJ è laboriosa; si stima che le macro risparmierebbero un ricercatore esperto fino a 15 min / NMJ speso per la segmentazione manuale dell&#39;immagine. Con uno o due decine di sinapsi valutati per ogni condizione o genotipo, questo riassume rapidamente fino a una considerevole quantità di tempo risparmiato, anche in studi su piccola scala. Quando si esegue grandi schermi, il guadagno di utilizzare alta analisi del throughput, rispetto alla valutazione manuale e la quantificazione, può essere immensa. Oltre a una maggiore produttività, le macro prontamente fornire un&#39;analisi obiettiva; escludono pregiudizi personali che altrimenti richiederebbero sperimentazioni in cieco nonché le differenze tra persone che si verificano quando più i ricercatori sono coinvolti nell&#39;analisi. Infine, le macro offrono un un sensibile e precisoANALISI DELLA caratteristiche NMJ, permettendo l&#39;identificazione di regolatori sinaptici che causano piuttosto sottile di drammatici difetti NMJ e rimaste sinora apprezzati dall&#39;occhio del ricercatore. Informazioni dettagliate sulle procedure di convalida e gli algoritmi utilizzati nelle macro si trovano nella pubblicazione Nijhof et al. 17. La funzionalità delle macro è stato validato per misurare in modo appropriato le caratteristiche morfologiche della Drosophila melanogaster NMJs al muscolo 4. Successivamente, è stato dimostrato che le macro sono stati anche adatto per analizzare le sinapsi in altri muscoli in questo organismo. È probabile che le macro possono essere utilizzati anche per misurare parametri morfologici di giunzioni neuromuscolari con struttura simile in altre specie, tra le altre specie Drosophila e altri insetti. Anche NMJs molto lontane nell&#39;evoluzione, per esempio, NMJs di topi, mostrano una conformazione strutturale abbastanza simile 29. Le macro non sono stati testati sui preparativi NMJ da altre specie, ma i potenziali utenti sono incoraggiati a testare le macro per tali scopi. E &#39;molto importante che l&#39;utente esplora le diverse auto-soglie e gli algoritmi per definire / scegliere le impostazioni macro più adatte per le immagini. Con queste impostazioni, una precisione di circa il 95% si ottiene quando la valutazione macro è stato confrontato con la valutazione manuale. Regolazione delle impostazioni macro correttamente segmento 100% delle immagini può essere una procedura molto laboriosa o addirittura impossibile. Pertanto, l&#39;esclusione di immagini non correttamente segmentati è raccomandato se il loro numero è inferiore al 5%. Evidentemente, se la qualità delle immagini è bassa, le macro si generano elevati rapporti di segmentazione di immagini insoddisfacenti. immagini di bassa qualità saranno allo stesso modo influenzare la valutazione manuale e possono quindi non essere legato alla performance delle macro. Tuttavia le macro sono piuttosto robusti come sono stati progettati per un&#39;immagines generata su un alto contenuto microscopio (un microscopio a fluorescenza automatico che permette l&#39;imaging di un gran numero di campioni) 17. Un punto critico è che l&#39;utente controlla visivamente tutte le immagini di risultati generati dalle macro. Questo permetterà di individuare ed escludere le immagini con la segmentazione insoddisfacente. Nella sezione 6 di questo protocollo, l&#39;utente viene guidato come regolare le impostazioni corrette segmentazione di immagini quando si esegue il sub-macro &quot;Analyse&quot;. Per conoscere rapidamente con i requisiti delle macro e come regolare le impostazioni macro una cartella chiamata &quot;Examples_adjusting impostazioni macro&quot; è incluso nel repository https://figshare.com/s/ec634918c027f62f7f2a macro. Tredici sottocartelle, ciascuna con immagini esempi ottenuti a piattaforme diverse microscopio (microscopi alto contenuto / confocale / fluorescenza) e diversi immunostainings, sono forniti. Un PDF intitolato “Esempi guida” è incluso nella stessacartella in cui vengono fornite le impostazioni necessarie per ogni esempio, insieme ad un documento di testo che fornisce i risultati attesi e risultati immagini. Le macro sono stati progettati per processare le immagini salvate come TIFF separati file, tuttavia alcuni utenti potrebbero aver salvato le loro immagini in un formato diverso. Il seguente sito web https://figshare.com/s/ec634918c027f62f7f2a 21 contiene una cartella denominata &quot;Drosophila NMJ&quot; dove tre file di esempio (esempio 1 - 3) e la &quot;Guida Esempi&quot; documento con le istruzioni dettagliate su come importare le immagini nella macro se non memorizzati come file separati .tiff si possono trovare anche nella stessa cartella. Insieme, &quot;Drosophila NMJ Morfometria&quot; e &quot;Drosophila NMJ Bouton morfometria&quot; macro quantificare dieci diverse caratteristiche NMJ: area NMJ, perimetro NMJ, numero di boutons, area bouton NMJ, lunghezza NMJ, giunzioni neuromuscolari più lunga di lunghezza ramo, numero di islands, numero di rami, il numero di punti di ramificazione e il numero di zone attive. Ciò fornisce un grande vantaggio rispetto gli strumenti finora disponibili che possono valutare solo uno o alcune caratteristiche sinaptiche 30, 31. Analisi quantitativa multiparametrica porta grande potenziale per nuove scoperte, ad esempio, per identificare nuovi regolatori che controllano uno fino a numerosi aspetti della biologia sinapsi. Esso fornisce anche la risoluzione necessaria per identificare i geni che coregulate esattamente uguali o sovrapposti caratteristiche NMJ e quindi suscettibili di operare in percorsi molecolari comuni. Infine, si apre la possibilità di studiare come diversi parametri sinaptiche correlazione tra loro in condizioni inalterate 17 e quali geni garantiscono tali correlazioni morfometrici coordinati. Nel loro insieme, questo protocollo illustra come utilizzare i due macro &quot;Drosophila NMJ Morfometria&quot; e&quot;Drosophila NMJ Bouton Morfometria&quot;, che svolgono quantificazione oggettiva e sensibile di dieci caratteristiche morfologiche NMJ in modo ad alta produttività.

Disturbi cognitivi come la disabilità intellettiva, disturbo dello spettro autistico e la schizofrenia sono spesso caratterizzati da anormale funzione sinaptica 1, 2, 3. Sinapsi morfologia e funzione sono strettamente intrecciati; difetti morfologici possono causare malfunzionamenti sinaptica e, inversamente, trasmissione sinaptica aberrante influenzeranno maturazione sinaptica e morfologia 4, 5, 6. Un certo numero di organismi modello sono state impiegate al fine di comprendere meglio la biologia delle sinapsi e far luce su come i cambiamenti sinaptici influenzano la funzione del cervello in salute e malattia 7, 8, 9. Drosophila NMJ è un ampiamente studiato e ben stabilito modello in vivo per glutamatergici synapse biologia 10, 11. Negli ultimi decenni, questo modello è stato utilizzato per studi fisiologici e gene-concentrata, così come per gli schermi genetici su larga scala, con lo scopo di rilevare le differenze morfologiche tra NMJs. In particolare, gli schermi genetici in avanti hanno identificato molti regolatori cruciali e meccanismi alla base dello sviluppo sinapsi e funzionamento 12, 13, 14, 15, 16. Tuttavia, la maggior parte di questi schermi invocati valutazione visiva del NMJ morfologia del terminale e sulla determinazione qualitativa delle anomalie sinaptiche o tracciatura semi-quantitativa di alcune caratteristiche morfologiche. Di conseguenza, piuttosto sottili anomalie morfologiche sinaptiche che non sono evidenti per l&#39;occhio umano potrebbero passare facilmente inosservate. Per essere in grado di rilevare completo differenze quantitative, leNMJ deve essere accuratamente valutata quantificando sistematica dei parametri morfologici di interesse. Misurare caratteristiche NMJ manualmente è laboriosa, soprattutto quando ci sono diverse caratteristiche NMJ di interesse e / o durante l&#39;esecuzione di screening genetici su larga scala. Per supportare multiparametrica, high-throughput analisi morfologica e per conseguire quantificazione oggettiva, due macro &quot;Drosophila NMJ morfometria&quot; e &quot;Drosophila NMJ Bouton morfometria&quot; sono stati sviluppati 17. Entrambe le macro vengono eseguite in open source software di analisi di immagine Fiji 18, e possono quantificare entrambe le immagini confocale e nonconfocal. Misure &quot;Drosophila NMJ morfometria&quot; terminali NMJ immunostained con il disco postsinaptica marcatore larga 1 (Dlg-1) o il presinaptica perossidasi di rafano (HRP), co-etichettati con il marcatore Bruchpilot zona attiva (BPA). Si quantifica nove Parame morfologicaters (descritto di seguito): area NMJ, perimetrali NMJ, numero di boutons, lunghezza giunzioni neuromuscolari, giunzioni neuromuscolari più lunga lunghezza ramo, numero di isole, numero di rami, il numero di punti di ramificazione e il numero di zone attive nel terminale sinaptico (Figura 1) . Anche se è presente un algoritmo per determinare il numero di boutons in questa macro, esso non soddisfa i criteri per la precisione 17. Per valutare correttamente il numero di boutons, è necessario utilizzare la macro &quot;Drosophila NMJ Bouton Morfometria&quot;, che è specificamente progettato per quantificare boutons su preparazioni NMJ immunostained da anti-sinaptotagmina (Syt) o della proteina stringa anti-cisteina (CSP), e co-immunolabeled con Brp. La macro &quot;Drosophila NMJ Bouton Morfometria&quot; quantifica i seguenti parametri: numero di boutons, NMJ zona bouton, lunghezza giunzioni neuromuscolari, giunzioni neuromuscolari maggiore lunghezza ramo, il numero di isole, il numero di rami, il numero di punti di ramificazione e il numero di zo attivonna (Figura 2). Le macro sono costituite da 3 sub-macro: (I) &quot;Convertire a impilare&quot; identifica tutti i file di immagine disponibili e crea Z-hyperstacks e proiezioni di massima intensità di entrambi i canali. Come uscita, questa macro genera due nuovi file ogni sinapsi chiamato &quot;stack_image_name&quot; e &quot;flatstack_image_name&quot;. II) &quot;Definire ROI&quot; si aprirà tutte le immagini di proiezione massime &quot;flatstack_image_name&quot; consecutivamente e presentarli con la richiesta di definire manualmente la regione di interesse (ROI) in cui è presente il terminale sinaptico specifico di interesse. Questo è stato realizzato per consentire esclusione delle sinapsi collegano ai muscoli adiacenti e / o altri tipi di terminali sinaptici (come 1s) che possono essere presenti nelle immagini 11. (III) &quot;Analyse&quot; applicato completamente automatizzato analisi per tutte le regioni delle immagini entro i confini della ROI. ComeA seguito di tale fase, l&#39;utente otterrà due nuovi file: &quot;results.txt&quot; dove verrà annotata tutta la misura numerica e un &quot;res_image_name.tif&quot; dove verranno illustrati i segmentazione di immagini sottostanti prodotte dalla macro. Durante l&#39;analisi dell&#39;immagine tre strutture sono derivati ​​da ogni terminale sinaptico: il contorno NMJ, lo scheletro giunzione neuromuscolare, e il numero di zone attive Brp-positivi. Lo schema NMJ viene utilizzato per determinare l&#39;area NMJ e il suo perimetro e una successiva separazione svolta fornisce il numero di boutons. Dallo scheletro, cinque funzioni NMJ si deducono: lunghezza totale NMJ, la somma della lunghezza del percorso continuo lungo collegare i due punti finali (maggiore lunghezza ramo), il numero di scomparti estranee al NMJ (denominate &quot;isole&quot; ), il numero di rami, e il numero di punti di ramificazione (un punto di diramazione collega tre o più rami). Il numero di zone attive è determinato nel Brp canale contandomacchie BRP-positivi. Lo schema annotato NMJ (linea gialla), lo scheletro NMJ (linea blu), e il numero di zone attive Brp-positivi (indicati da foci bianco) vengono visualizzati in un quadro risultati e le misurazioni dei parametri sono trattati per un (. txt) file di output (Figura 3). Drosophila NMJ Morfometria&quot; e &#39;Drosophila NMJ Bouton Morfometria&#39; sono stati descritti e ampiamente convalidati da Nijhof et al. 17. Questo manoscritto si concentra sulla metodologia per analizzare NMJ morfologia utilizzando le macro &#39;Drosophila NMJ morfometria&#39; e &#39;Drosophila NMJ Bouton morfometria&#39;. Tuttavia, prima di analisi macro-assistita, dissezioni NMJ e immunostainings devono essere eseguiti. Questi sono passi fondamentali, e la combinazione di marcatori utilizzati per immunoistochimica deve essere adatto per analisi macro. Questi passaggi sono brevemente menzionati in séction 1 di questo protocollo e dirigere l&#39;utente a riferimenti che descrivono in dettaglio i protocolli per eseguire queste procedure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunostainings</td>
			<td></td>
			<td></td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-discs large 1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AFFN-DLG1-4D6</td>
			<td>1/25 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)</td>
		</tr>
		<tr>
			<td>Rabbit anti-horseradish peroxidase</td>
			<td>Jackson IR</td>
			<td>323-005-021</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Synaptotagmin</td>
			<td>Gift from Hugo Bellen</td>
			<td></td>
			<td>Jan-00</td>
		</tr>
		<tr>
			<td>Mouse anti-Cysteine string protein</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>DCSP-1(ab49)</td>
			<td>1/10 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)&#160;</td>
		</tr>
		<tr>
			<td>Mouse anti-Bruchpilot</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Jan-50</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Life technologies</td>
			<td>A11029</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 568</td>
			<td>Life technologies</td>
			<td>A11011</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Zenon Alexa Fluor 568 Mouse IgG1 Labeling Kit</td>
			<td>ThermoFisher</td>
			<td>Z25006</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Confocal microscope or fluorescence microscope</td>
			<td>Leica SP5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio imager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Mac or Pc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two algorithms for high-throughput and multi-parametric quantification of drosophila neuromuscular junction morphology

morfologia sinaptica è strettamente correlata all&#39;efficacia sinaptica, e in molti casi difetti morfologici sinapsi in definitiva portare a malfunzionamenti sinaptica. Larvale Drosophila giunzione neuromuscolare (NMJ), un modello ben consolidato per sinapsi glutammatergica, è stato ampiamente studiato per decenni. Identificazione di mutazioni causano difetti morfologici NMJ rivelato un repertorio di geni che regolano lo sviluppo delle sinapsi e funzione. Molti di questi sono stati identificati in studi su larga scala che si è concentrato su approcci qualitativi per rilevare anomalie morfologiche della Drosophila NMJ. Un inconveniente di analisi qualitative è che molti giocatori sottili che contribuiscono alla morfologia NMJ probabile rimangono inosservati. Considerando analisi quantitative sono necessarie per rilevare le differenze morfologiche sottili, tali analisi non sono ancora comunemente eseguite perché sono laboriose. Questo protocollo descrive in dettaglio due algoritmi di analisi dell&#39;immagine &quot;Drosophila </em&gt; NMJ Morfometria&quot; e &#39;Drosophila NMJ Bouton Morfometria&#39;, disponibile come macro compatibili Fiji, per l&#39;analisi quantitativa, precisa e oggettiva morfometrica della Drosophila NMJ. Questa metodologia è sviluppato per analizzare terminali NMJ immunolabeled con i marcatori comunemente utilizzati Dlg-1 e . BRP Inoltre, la sua più ampia applicazione ad altri marcatori, come Hrp, Csp e Syt è presentato in questo protocollo Le macro sono in grado di valutare nove caratteristiche NMJ morfologiche:. Area NMJ, perimetro NMJ, numero di boutons, lunghezza NMJ, NMJ più lungo ramo lunghezza, numero di isole, il numero di rami, il numero di punti di ramificazione e il numero di zone attive nel terminale NMJ.

Watch this Scientific Journal Video about Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology at JoVE.com

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Sono stati creati due algoritmi di analisi dell&#39;immagine, &quot;Drosophila NMJ morfometria&quot; e &quot;Drosophila NMJ Bouton morfometria&quot;, quantificare automaticamente nove caratteristiche morfologiche della giunzione neuromuscolare Drosophila (NMJ).

Due Algoritmi per high-throughput e multi-parametrica Quantificazione degli<em&gt; Drosophila</em&gt; Giunzione neuromuscolare Morfologia

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

two-algorithms-for-high-throughput-multi-parametric-quantification

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

9.6K Views.  Radboud University Medical Center.  Sono stati creati due algoritmi di analisi dell'immagine, "Drosophila NMJ morfometria" e "Drosophila NMJ Bouton morfometria", quantificare automaticamente nove caratteristiche morfologiche della giunzione neuromuscolare Drosophila (NMJ). 

Video: Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

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.

Dissezione e immagini di zone attive nel Drosophila Giunzione neuromuscolare

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

Ricerca

Neuroscienze

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Dissezione del muscolo Transversus Abdominis per l'analisi della giunzione neuromuscolare a montaggio intero

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Rilevamento di<em&gt; In Situ</em&gt; Complessi proteina-proteina del<em&gt; Drosophila</em&gt; Larvale neuromuscolare Junction Utilizzando prossimità legatura Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

FIM Imaging e FIMtrack: due nuovi strumenti che ad alta produttività e costo effettivo Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

High Throughput test per esaminare Preferenze deposizione delle uova dei singoli<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Perché Quantificazione Matters: Caratterizzazione dei fenotipi al<em&gt; Drosophila</em&gt; Larvale Giunzione neuromuscolare

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Funzionalità di misurazione della giunzione neuromuscolare

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

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