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>

<ol>
	<li>Zhang, B., Freeman, M. R., Waddell, S. <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 neurobiology: a laboratory manual. , (2010).</li><li>Frank, C. A., 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=New+approaches+for+studying+synaptic+development,+function,+and+plasticity+using+Drosophila+as+a+model+system.">New approaches for studying synaptic development, function, and plasticity using Drosophila as a model system.</a> J Neurosci. 33, 17560-17568 (2013).</li><li>Mudher, A., Newman, T. <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 : a toolbox for the study of neurodegenerative disease. , (2008).</li><li>Bilen, J., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+human+neurodegenerative+disease.">Drosophila as a model for human neurodegenerative disease.</a> Annu Rev Genet. 39, 153-171 (2005).</li><li>Jakubowski, B. R., Longoria, R. A., Shubeita, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+throughput+and+sensitive+method+correlates+neuronal+disorder+genotypes+to+Drosophila+larvae+crawling+phenotypes.">A high throughput and sensitive method correlates neuronal disorder genotypes to Drosophila larvae crawling phenotypes.</a> Fly (Austin). 6, 303-308 (2012).</li><li>Caldwell, J. C., Miller, M. M., Wing, S., Soll, D. R., Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+larval+locomotion+in+Drosophila+chordotonal+organ+mutants).">Dynamic analysis of larval locomotion in Drosophila chordotonal organ mutants).</a> Proc Natl Acad Sci U S A. 100, 16053-16058 (2003).</li><li>Jahn, T. R., 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=Detection+of+early+locomotor+abnormalities+in+a+Drosophila+model+of+Alzheimer's+disease.">Detection of early locomotor abnormalities in a Drosophila model of Alzheimer's disease.</a> J Neurosci Methods. 197, 186-189 (2011).</li><li>Donelson, N. C., 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=High-resolution+positional+tracking+for+long-term+analysis+of+Drosophila+sleep+and+locomotion+using+the+&amp;#34;tracker&amp;#34;+program.">High-resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the &amp;#34;tracker&amp;#34; program.</a> PLoS ONE. 7, e37250 (2012).</li><li>Slawson, J. B., Kim, E. Z., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+video+tracking+of+locomotion+in+adult+Drosophila+melanogaster.">High-resolution video tracking of locomotion in adult Drosophila melanogaster.</a> J Vis Exp. (24), (2009).</li><li>Colomb, J., Reiter, L., Blaszkiewicz, J., Wessnitzer, J., Brembs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+tracking+and+analysis+of+adult+Drosophila+locomotion+in+Buridan's+paradigm+with+and+without+visual+targets.">Open source tracking and analysis of adult Drosophila locomotion in Buridan's paradigm with and without visual targets.</a> PLoS ONE. 7, e42247 (2012).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1965).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> J Insect Physiol. 46, 439-442 (2000).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly pushing : the theory and practice of Drosophila genetics.. , (2004).</li><li>Duncan, J. E., Lytle, N. K., Zuniga, A., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microtubule+Regulatory+Protein+Stathmin+Is+Required+to+Maintain+the+Integrity+of+Axonal+Microtubules+in+Drosophila.">The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila.</a> 8, e683244 (2013).</li><li>Belmont, L. D., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+that+interacts+with+tubulin+dimers+and+increases+the+catastrophe+rate+of+microtubules.">Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.</a> Cell. 84, 623-631 (1996).</li><li>Cassimeris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncoprotein+18/stathmin+family+of+microtubule+destabilizers.">The oncoprotein 18/stathmin family of microtubule destabilizers.</a> Curr Opin Cell Biol. 14, 18-24 (2002).</li></ol>

Gli autori hanno dichiarato che non esistono interessi in competizione.

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trinocular Stereozoom Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ6145TR</td>
			<td>&#189; 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 (43mm thread)</td>
			<td>Qioptiq Imaging Solutions</td>
			<td>25-70-10-02</td>
			<td></td>
		</tr>
		<tr>
			<td>58mm to 48mm Step Down Ring</td>
			<td>B&#38;H Video</td>
			<td>GBSDR5848</td>
			<td></td>
		</tr>
		<tr>
			<td>48mm to 43mm Step Down Ring</td>
			<td>B&#38;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>1.5ml Spectroscopic PolystereneCuvette</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

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

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

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

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

Visualizzazione dei nervi larvali segmentale in 3 Rd Instar Drosophila I preparativi larvali

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

Ricerca

Neuroscienze

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

Mappatura e applicazione di Enhancer-trappola Espressione Flippase in larvali e adulti Drosophila SNC

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Dissezione e immunoistochimica di larvale, pupa e adulto<em&gt; Drosophila</em&gt; Retine

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Analisi funzionale del circuito di alimentazione larvale in Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Immagini dal vivo di<em&gt; Drosophila</em&gt; larvali neuroblasti

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

Measuring the impacts of environmental contaminants on fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints. We describe methods including video technology to quantify swimming behavior of early life stage white sturgeon (Acipenser transmontanus) during and after 96 hr acute exposures to various concentrations of copper.

Quantificare Pesce Nuoto Comportamento in risposta all&#39;esposizione acuta acquosa di rame utilizzando Computer Assisted Video e Digital Image Analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Comportamenti olfattive analizzati da Computer di monitoraggio di<em&gt; Drosophila</em&gt; In un Olfattometro quattro quadranti

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Gusto Preferenza Assay per adulti<em&gt; Drosophila</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Rimozione di<em&gt; Drosophila</em&gt; Tessuto muscolare da larvali Filetti per l&#39;analisi di immunofluorescenza dei neuroni sensoriali e cellule epidermiche

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilizzando canali lineari Agarose per studiare<em&gt; Drosophila</em&gt; Comportamento larvale Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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Visualizzazione dei nervi larvali segmentale in 3^Rd Instar Drosophila I preparativi larvali

Mappatura e applicazione di Enhancer-trappola Espressione Flippase in larvali e adulti Drosophila SNC

Dissezione e immunoistochimica di larvale, pupa e adulto

Analisi funzionale del circuito di alimentazione larvale in Drosophila

Immagini dal vivo di

Quantificare Pesce Nuoto Comportamento in risposta all'esposizione acuta acquosa di rame utilizzando Computer Assisted Video e Digital Image Analysis

Comportamenti olfattive analizzati da Computer di monitoraggio di

Gusto Preferenza Assay per adulti

Rimozione di

Utilizzando canali lineari Agarose per studiare