This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1. fissare la testina sui collari e inclusione in paraffina Nota: Tutte le fasi del processo di fissazione dovrebbe essere fatto in una cappa aspirante. Benzoato di metile, anche se non presentano un rischio per la salute, ha un odore molto distinta, che può essere schiacciante, se non gestita in una cappa aspirante. <ol><li> Prima anestetizzare le mosche, portare 50 mL di soluzione di Carnoy aggiungendo 15 ml di cloroformio e 5 ml di acido acetico glaciale a 30 ml di 99% di etanolo (non mescolare cloroformio e acido acetico). Versare in un contenitore di vetro con un fondo piatto, come un piatto di cristallizzazione, il garantire che i collari possono distesi e sono completamente coperti dalla soluzione. </li><li> Anestetizzare le mosche con CO 2 o etere. </li><li> mosche filo (fino a 20) con la maggior parte collari per il collo nelle collari che utilizzano pinze. Ricordare per allineare tutte le teste con lo stesso orientamento, come mostrato in figura 1A, ed essere gentile per garantire che nessun danno si verifica alla testa o occhi. </li><li> Includere mosche sine Oculis (frecce, Figura 1A) in posizioni casuali in modo che l&#39;ordine delle mosche può essere facilmente identificato in sezioni. Inoltre, se le mosche sperimentali hanno un colore degli occhi chiaro o bianco, filo alcune mosche dagli occhi rossi, come il tipo selvaggio, tra di loro per garantire che il pigmento è considerata sufficiente a macchiare la diapositiva. Registrare l&#39;ordine delle mosche su un foglio protocollo nonché il numero collare se si utilizza più di un collare. </li><li> Una volta che un collare è stato finito, posizionarlo nella soluzione Carnoy preparato per 3,5-4 ore. </li><li> Dump la soluzione Carnoy nel contenitore di smaltimento appropriato e iniziare i lavaggi etanolo. Assicurarsi di versare lentamente in modo da non disturbare il posizionamento dei collari nel contenitore. </li><li> Lavare i collari per 30 minuti in 99% etanolo due volte. </li><li> Lavare i collari in 100% di etanolo per 1 h. Assicurarsi di modificare i lavaggi in tempo per evitare overdehydration. </li><li> Mettere i collari di benzoato di metileO / N a temperatura ambiente. Sigillare il contenitore con parafilm per impedire l&#39;evaporazione del benzoato di metile. </li><li> Versare il methylbenozate nel corretto contenitore monouso nella cappa. Aggiungere una miscela precedentemente preparata di 1: 1 a basso punto di fusione (56-57 ° C) cera di paraffina e benzoato di metile. Da questo punto in poi, i collari devono essere tenuti in un incubatore a 65 ° C per assicurarsi che la paraffina non si indurisce. </li><li> Versare il methylbenozate e la miscela di paraffina nel giusto contenitore monouso, e versare fuso paraffina pura, mantenuta a 65 ° C, sui collari. </li><li> Modificare la paraffina dopo 30 minuti e ripetere questo almeno 5 volte. Almeno 6 - 8 lavaggi devono essere eseguiti. </li><li> Una volta che i lavaggi sono completi, inserire i collari in una vaschetta per il ghiaccio in gomma con scanalature circa le dimensioni dei collari. Versare paraffina fusa su di loro fino a quando completamente ricoperta e lasciarlo indurire O / N (cercare di evitare bolle d&#39;aria). </li><li> Rimuovere i blocchi di paraffina containing i collari dal vassoio del cubo di ghiaccio. Separare il blocco di paraffina dal collare con una lama di rasoio, rompendo delicatamente il collare. Le teste saranno nel blocco di paraffina mentre i corpi rimarranno nel collare. I blocchi possono essere conservati a temperatura ambiente. </li><li> Per pulire i collari, ammollo in un agente deparafinization a 65 ° C per rimuovere la paraffina, pulito, con lavaggio luce, e lavare in etanolo prima di riutilizzarlo. </li></ol> 2. Sezioni e montaggio <ol><li> Riscaldare una piastra riscaldante a 50 ° C. Posizionare i titolari oggetto (o blocchi di montaggio in metallo) e lame di rasoio sul piatto e lasciarli riscaldare. </li><li> A seconda dell&#39;orientamento desiderato per il sezionamento, collegare il blocco di paraffina o con le teste verso il lato (per sezioni orizzontali) o verso l&#39;alto (per le sezioni frontali) a un blocco di montaggio riscaldata (fusione brevemente il blocco sul lato di contatto). Rimuovere il blocco dalla piastra di riscaldamento e lasciarlo raffreddare per almeno 10 min garantire che la paraffina è indurito sufficiente per una buona tenuta sul blocco di montaggio. Mantenere la fila di teste allineate parallelamente alla superficie del blocco il più possibile per evitare sezioni irregolari. </li><li> Prendere una lametta e tagliare l&#39;eccesso di paraffina dalle teste volo in modo che solo una piccola fila con le teste incorporate rimane (la lama può essere riscaldato per facilitare il taglio). Assicurarsi di non tagliare troppo in modo che la paraffina non si rompa durante il sezionamento (più il taglio può essere fatto durante il sezionamento). </li><li> Posizionare il blocco di montaggio nel supporto oggetto del microtomo e garantire che l&#39;allineamento della fila di teste è più possibile parallelo al bordo della lama. </li><li> Preparare vetrini da microscopio coprendole con un sottile strato di soluzione di poli-L-lisina (PLL) e lasciate asciugare per 5 min. Coprire con acqua poco prima di utilizzare. </li><li> Tagliare 7 sezioni micron e trasferire il nastro delle sezioni alla slitta galleggiante sull&#39;acqua. &lt;br /&gt; Nota: Per ottenere l&#39;intero cervello per le sezioni orizzontali, raccogliamo il nastro da quando si inizia a tagliare in un occhio fino a quando la testa è stata completamente tagliato (il taglio dal proboscide nel cervello). Più di una diapositiva può essere necessaria per l&#39;intera testa. </li><li> Posizionare il vetrino su una piastra di calore a 37 ° C e permettere al nastro di espandersi per circa 1 min. </li><li> Rimuovere l&#39;acqua in eccesso (versando fuori o utilizzando un tessuto) e asciugare il diapositive O / N. </li><li> Rimuovere la cera di paraffina dal vetrino posizionando i vetrini in un uomo alto, vaso slide-colorazione verticale riempito con un agente deparafinization (che copre completamente le sezioni). Effettuare 3 lavaggi di 30 min - 60 min ciascuno. </li><li> Rimuovere il vetrino dal lavaggio finale. Mettere 2 gocce di media incorporamento sul vetrino e coprirlo con un largo coprioggetti. </li></ol> 3. Fotografare e l&#39;analisi dei Sezioni <ol><li> Lasciare i vetrini preparati ad asciugare per 1 - 2 d. Poi, li esaminerà in una microsc a fluorescenzaope sotto la luce blu. </li><li> Utilizzare un ingrandimento minore per determinare l&#39;orientamento delle mosche e per individuare la zona di interesse se concentrandosi su una regione specifica. NOTA: Per le mosche SWS (vedi figura 2), troviamo la sezione che contiene il grande commessura e scattare una foto (di solito a 40X di ingrandimento). Analizzando l&#39;intero cervello (come in figura 3), si scorrere tutte le sezioni da una testa e sia fotografare la sezione con il fenotipo più grave o tutte le sezioni che mostrano vacuoli. </li><li> Per un&#39;analisi doppio cieco, prendere e immagini numero senza conoscere il genotipo e registrare il numero di collare e la posizione della testa della fila di identificarli in seguito. </li><li> Una volta che sono state prese le immagini, analizzarli utilizzando un software di imaging. </li><li> Contare il numero di vacuoli per sezione o per capo. Per misurare la dimensione vacuolo, aprire le immagini in un programma software e selezionare i vacuoli con una selezione troppol. Determinare la quantità di pixel nei vacuoli selezionati. </li><li> Per la conversione di micron 2, scattare una foto di uno scivolo di calibrazione micrometrico al ingrandimento utilizzato per l&#39;acquisizione di foto. Determinare la quantità di pixel in 100 micron 2 per calcolare il fattore di conversione. </li><li> Convertire il numero totale di pixel in micron 2 dividendo il numero di pixel per il fattore di conversione calcolata nel passaggio precedente. </li></ol>

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocerebrosidase+Deficiency+in+Drosophila+Results+in+alpha-Synuclein-Independent+Protein+Aggregation+and+Neurodegeneration.">Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration.</a> PLoS Genet. 12, e1005944 (2016).</li><li>Dias-Santagata, D., Fulga, T. A., Duttaroy, A., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+mediates+tau-induced+neurodegeneration+in+Drosophila.">Oxidative stress mediates tau-induced neurodegeneration in Drosophila.</a> J Clin Invest. 117, 236-245 (2007).</li><li>Kretzschmar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+and+neuronal+expression+of+polyglutamine+proteins+induce+behavioral+changes+and+aggregate+formation+in+Drosophila.">Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila.</a> Glia. 49, 59-72 (2005).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+for+TDP-43+proteinopathy.">A Drosophila model for TDP-43 proteinopathy.</a> Proc Natl Acad Sci U S A. 107, 3169-3174 (2010).</li></ol>

Gli autori non hanno nulla da rivelare.

Il metodo descritto fornisce un mezzo per quantificare neurodegenerazione nel cervello di Drosophila. Mentre altri metodi, come conteggio di un tipo di cellula specifico, possono essere utilizzati per identificare neurodegenerazione, il vantaggio di questo metodo è che può essere applicata più in generale. Contare le cellule richiede che queste cellule possono essere identificate in modo affidabile utilizzando un anticorpo specifico o l&#39;espressione di un marcatore specifico per cellule, che non è sempre...

Con l&#39;aumento dell&#39;aspettativa di vita, malattie neurodegenerative come il morbo di Alzheimer o di Parkinson sono diventati una minaccia per la salute crescente per la popolazione generale. Secondo il National Institutes of Health, 115 milioni di persone nel mondo sono previsti per essere colpiti da demenza nel 2050. Anche se sono stati compiuti progressi significativi per identificare geni e fattori di rischio coinvolti in almeno alcune di queste malattie, per molti di loro, il sottostante meccanismi molecolari sono ancora sconosciuti o non ben compreso. Semplici invertebrati organismi modello come Caenorhabditis elegans e Drosophila melanogaster offrono una varietà di vantaggi sperimentali per studiare i meccanismi di malattie neurodegenerative, tra cui un breve ciclo di vita, gran numero di discendenti, e la disponibilità di metodi ben consolidati e, a volte unici genetici e molecolari 1 -12. Inoltre, questi organismi sono suscettibili di imparzialeschermi di interazione in grado di identificare i fattori che contribuiscono a queste malattie per i loro effetti aggravanti o Ameliorating sui fenotipi neurodegenerative. Analizzando tali interazioni genetiche e valutare gli effetti dell&#39;invecchiamento richiede protocolli quantitativi per rilevare neurodegenerazione e misurare la sua gravità. Questa valutazione può essere fatta in modo relativamente facile quando si misura aspetti comportamentali di Drosophila, quali l&#39;apprendimento olfattivo, geotassi negativi, o fototassi veloce, che forniscono un valore numerico delle prestazioni 13-21. È anche possibile determinare gli effetti sulla sopravvivenza neuronale contando neuroni. Tuttavia, questo è possibile solo quando concentrandosi su una specifica popolazione che è chiaramente identificabile, come i neuroni dopaminergici che sono interessati nel PD, e anche allora, i risultati sono stati controversi 22-24. Il protocollo qui descritto utilizza il metodo collare per eseguire sezioni seriali paraffina, un metodoche è stato originariamente sviluppato da Heisenberg e Böhl, che lo utilizzò per isolare i mutanti cerebrali anatomiche in Drosophila 25. L&#39;utilizzo del metodo collare è stato successivamente adattato, anche in criosezioni, vibratome sezioni e profilati in plastica 26-28. Ecco, questo metodo viene utilizzato per ottenere sezioni seriali di tutta la testa mosca, che può quindi essere utilizzato per misurare i vacuoli che si sviluppano in mosche con fenotipi neurodegenerative 16,21,29-32. Queste misurazioni possono essere eseguite in specifiche aree cerebrali o in grado di coprire l&#39;intero cervello; il secondo approccio permette di identificare anche fenotipi degenerative deboli, come osservato durante l&#39;invecchiamento. Infine, quando si utilizzano i collari, fino a 20 in linea può essere elaborato come una preparazione, che è non solo meno tempo, ma consente anche per l&#39;analisi di controllo e mosche sperimentali nella stessa preparazione, minimizzando artefatti da lievi variazioni la preparazione.

<table><tbody><tr><td>Name of the Reagent/Equipment</td><td>Company</td><td>Catalog Number</td><td></td></tr><tr><td>Collar</td><td>Genesee Scientific</td><td>TS 48-100</td><td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A description on how to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&amp;nbsp;</td></tr><tr><td>Rubber ice cube tray for embedding</td><td>Household store</td><td></td><td>The size can be made-to-fit by glueing in additional walls&amp;nbsp;</td></tr><tr><td>Crystallizing dish</td><td>Fisher Scientific company</td><td>08-762-3</td><td></td></tr><tr><td>Ether</td><td>Fisher Scientific Company</td><td>E138-1</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories Inc.</td><td>2701</td><td></td></tr><tr><td>Choloroform</td><td>Fisher Scientific Company</td><td>C298-500</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher Scientific Company</td><td>A38-212</td><td></td></tr><tr><td>Methylbenzoate</td><td>Fisher Scientific Company</td><td>M205-500</td><td>Distinct Odor - Use in fume hood!</td></tr><tr><td>Low Melting Point Paraffin Wax</td><td>Fisher Scientific Company</td><td>T565</td><td>Make sure to keep extra melted in a 65&amp;deg;C waterbath</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>Reichert Jung 2040 Autocut</td><td></td></tr><tr><td>Microscope Slide</td><td>Fisher Scientific Company</td><td>12-550D</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific Company</td><td>12-545-M</td><td></td></tr><tr><td>SafeClear</td><td>Fisher Scientific Company</td><td>314-629</td><td>Three different containers for washes</td></tr><tr><td>Vertical Staining Jar with Cover</td><td>Ted Pella Inc.&amp;nbsp;</td><td>432-1</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific Company</td><td>SP15-500</td><td></td></tr><tr><td>Poly-L-Lysine Solution (PLL)</td><td>Sigma Life Science</td><td>P8290-500</td><td></td></tr></tbody></table>

mass histology to quantify neurodegeneration in drosophila

malattie neurodegenerative progressive come la malattia di Alzheimer (AD) o malattia di Parkinson (PD) è una crescente minaccia per la salute umana in tutto il mondo. Sebbene i modelli di mammiferi hanno fornito importanti informazioni sui meccanismi alla base della patogenicità, la complessità dei sistemi di mammiferi insieme con i loro alti costi stanno limitando il loro utilizzo. Pertanto, il semplice ma ben consolidata modello di sistema Drosophila fornisce un&#39;alternativa per lo studio dei meccanismi molecolari che sono colpite in queste malattie. Oltre a deficit comportamentali, malattie neurodegenerative sono caratterizzate da fenotipi istologici come la morte neuronale e assonopatia. Per quantificare la degenerazione neuronale e per determinare come esso è influenzato da fattori genetici e ambientali, usiamo un approccio istologico che si basa sulla misurazione dei vacuoli nel cervello mosca adulta. Per ridurre al minimo gli effetti di errore sistematico e di confrontare direttamente le sezioni di controllo e expmosche erimental in una preparazione, si usa il metodo &#39;collare&#39; per le sezioni di paraffina. Neurodegeneration viene valutata misurando la dimensione e / o il numero di vacuoli che si sono sviluppate nel cervello mosca. Questo può essere fatto sia concentrandosi su una specifica regione di interesse o analizzando l&#39;intero cervello ottenendo sezioni seriali che attraversano la testa completa. Pertanto, questo metodo permette di misurare non solo la degenerazione grave, ma anche fenotipi relativamente lievi che sono rilevabili solo in alcune sezioni, come avviene durante il normale invecchiamento.

Utilizzando i risultati metodo descritto in sezioni seriali colorati con l&#39;occhio del pigmento 33 che comprendono l&#39;intera testa mosca. Una parte di questo è mostrato in Figura 1B, dove le sezioni da una testa individuo sono mostrati dall&#39;alto verso il basso. Le sezioni di diverse mosche sono visti da sinistra a destra in questo esempio. Per facilitare l&#39;orientamento e l&#39;identificazione delle mosche, una mosca senza occhi (Oculis sinus...

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila è ampiamente usato come sistema modello per studiare neurodegenerazione. Questo protocollo descrive un metodo con cui la degenerazione, come determinato dalla formazione vacuolo nel cervello, può essere quantificato. Si riduce anche effetti dovuti alla procedura sperimentale di elaborazione e controllo sezionamento e mosche sperimentali come un campione.

Messa Istologia per quantificare neurodegenerazione in<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

10.1K Views.  Oregon Health & Sciences University.  Drosophila è ampiamente usato come sistema modello per studiare neurodegenerazione. Questo protocollo descrive un metodo con cui la degenerazione, come determinato dalla formazione vacuolo nel cervello, può essere quantificato. Si riduce anche effetti dovuti alla procedura sperimentale di elaborazione e controllo sezionamento e mosche sperimentali come un campione. 

Video: Mass Histology to Quantify Neurodegeneration in Drosophila

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Deficit Locomotore dosaggio, apprendimento e memoria in Drosophila Modelli di neurodegenerazione

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Ricerca

Neuroscienze

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In Vivo Avanti Schermo Genetico per Identificare Nuovi Geni Neuroprotettivi in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Genetica

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissezione e macchiatura immunofluorescente del corpo di fungo e del fotoricettore neuroni nel cervello adulto Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Biologia dello sviluppo

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Valutare fenotipi neurodegenerative<em&gt; Drosophila</em&gt; Neuroni dopaminergici mediante saggi arrampicata e Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

Una semplice metodologia di lesioni meccaniche neuronali da studiare<em&gt; Drosophila</em&gt; Degenerazione dei motori neuronali

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Biologia cellulare quantitativa della neurodegenerazione in Drosophila attraverso analisi imparziale delle proteine fluorescente contrassegnate usando ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Valutazione morfologica e funzionale degli assoni e delle loro sinapsi durante la morte di Axon in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

Visualizzazione della degenerazione sinaptica nella Drosophila adulta in associazione con la neurodegenerazione

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression patterns. Traditional methods often involve challenges such as performing complex dissection, maintaining tissue integrity, and visualizing specific expression patterns during high-resolution imaging. We present an optimized protocol that combines cryosectioning with fluorescence staining and immunostaining. This method improves tissue preservation and signal clarity and reduces the need for laborious dissection for Drosophila brain imaging. The method entails rapid dissection, optimal fixation, cryoprotection, and cryosectioning, followed by fluorescent staining and immunostaining. The protocol significantly reduces tissue damage, enhances antibody penetration, and yields sharp, well-defined images. We demonstrate the effectiveness of this approach by visualizing specific neural populations and synaptic proteins with high fidelity. This versatile method allows for the analysis of various protein markers in the adult brain across multiple z-planes and can be adapted for other tissues and model organisms. The protocol provides a reliable and efficient tool for researchers conducting high-quality immunohistochemistry in Drosophila neurobiology studies. This method's detailed visualization facilitates comprehensive analysis of neuroanatomy, pathology, and protein localization, making it particularly valuable for neuroscience research.

This study presents a simplified protocol for tissue processing involving decapitation, fixation, cryosectioning, fluorescence staining, immunostaining, and imaging, which can be extended to confocal and multiphoton imaging. The method maintains efficacy comparable to complex dissections, bypassing the need for advanced motor skills. Quantitative image analysis provides extensive investigative potential.

Criosezione diretta delle teste di Drosophila per una migliore colorazione a fluorescenza cerebrale e immunocolorazione

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression ...

Assessing the Structural Integrity of the Dorsal Longitudinal Muscle Neuromuscular Junctions in Drosophila

Source: Sidisky, J. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61363">Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration.</a> J. Vis. Exp. (2020).
This video demonstrates a protocol for assessing the structural integrity of neuromuscular junctions in the dorsal longitudinal muscles of Drosophila. The procedure involves staining neurons and muscles with two distinct antibodies and comparing fluorescence patterns between mutant and wild-type flies. A reduction in neuronal staining in mutant flies indicates neurodegeneration, while the muscle structure remains intact.

Valutazione dell'integrità strutturale delle giunzioni neuromuscolari muscolari longitudinali dorsali in Drosophila

1. Flash freezing and thorax bisection

	Before beginning the bisections, fill a Dewar flask with liquid nitrogen wearing proper cryo-protective gloves ...

Messa Istologia per quantificare neurodegenerazione in

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