Questo lavoro è stato sostenuto dal National Institutes of concede Salute RO1GM-33205 e MH-067284-LC Griffith e da una borsa di studio estiva Brandeis di ricerca dell&#39;Università di laurea a New Jersey Hornstein. Esperimenti preliminari per questa tecnica sono stati eseguiti presso il Marine Biological Laboratory come parte del 2008 e l&#39;estate dei sistemi neurali corso Behavior (NIMH sovvenzione: R25 MH059472) in Woods Hole, Massachusetts.

Parte 1: la cura degli animali e croci genetica <ol><li> Mantenere UAS-ChR2 e OK371-GAL4 linee di volare in bottiglie separate contenenti supporti standard 8 volare. </li><li> Raccogliere vergini di OK371-GAL4 linee volo ed i maschi di UAS-ChR2 linee. </li><li> Mettere maschi e femmine in una fiala contenente dei media volare miscela standard con 1 mM tutto-trans retinico (ATR). ATR cibo dovrebbe essere presa da primo media volare fusione regolare in un forno a microonde per circa 1 minuto. Una volta sciolto, lasciare raffreddare per circa 30 secondi a un minuto e poi aggiungere 100 ml di 100 mM ATR in 100 ETOH% per ogni 10 ml di media volare. Fiale luogo il ghiaccio, quindi memorizzare in una zona al buio a 4 ° C. </li><li> Lascia compagno di mosche e depongono le uova in una zona al buio a 22-25 ° C. </li><li> Attendere 3-4 giorni fino al 3 larve instar sono visibili, a quel punto, disporre di mosche adulte. </li></ol> Parte 2: configurazione Rig <ol><li> Allegare eventuali 10x dissezione oculare portata (nel nostro caso, da Carl Zeiss Inc., www.zeiss.com, Thornwood, NY), al blu sorgente luminosa a LED con dissipatore di calore (laboratori di Thor, www.thorlabs.com, Newton, NJ ). Fissare a base magnetica con post e morsetto. Coprire dissipatore con uno schermo a massa per ridurre il rumore elettrico generato dalla sorgente luminosa. </li><li> Luogo base magnetica con sorgente di luce sul tavolo aria di elettrofisiologia rig. </li><li> Collegare la fonte di luce per il controllo del circuito e collegare al circuito di controllo di tensione esterna (Powerlab 4 / 30, ADInstruments, www.adinstruments.com, Colorado Springs, CO). </li><li> Dare impulsi di 1-5 V per circuito di controllo per attivare la luce blu. </li><li> Regola base magnetica e la fonte di luce fino a fascio di luce blu è focalizzata sulla zona da occupata da dissezione larvale. </li></ol> Parte 3: Dissection <ol><li> Luogo sei pin 0,1 millimetri di insetti sul pavimento di una sylgard alberato piatto. </li><li> Rimuovere un 3 ° larve instar dai media cibo e il luogo in qualsiasi plastica Petri-piatto. </li><li> Sciacquare larve con soluzione salina per rimuovere gli alimenti. </li><li> Larve posto nella dissezione piatto vicino al pin tirato e aggiungere saline al livello ½. </li><li> Orientare le larve in modo che potete vedere due tubi argentati (trachea) che corre lungo la superficie dorsale dell&#39;animale. </li><li> Inserire un perno di grandi dimensioni direttamente in coda tra i tubi tracheali. Tenere le larve verso il basso e inserire un secondo perno di grandi dimensioni in testa, avendo cura di allungare il corpo in senso longitudinale. </li><li> Fai una piccola incisione vicino alla coda, e continuate in su la lunghezza del corpo. Assicurarsi che le punte delle forbici sono allevati in modo da non tagliare accidentalmente nervi ventrale e / o dei muscoli della parete del corpo. </li><li> Posto quattro perni sui quattro angoli del tronco dell&#39;animale ormai aperto. Metterli nel piatto dissezione in modo da raccordare l&#39;animale. Preparazione pin out insegnato. </li><li> Con pinze e forbici, togliere viscere dell&#39;animale, trachea e corpi grassi. </li><li> Sciacquare la preparazione con soluzione salina. </li><li> Individuare i lobi frontali (come si vede in Fig. A) e ganglio ventrale della preparazione. </li><li> Utilizzando micro-forbici, tagliare con attenzione attraverso il ganglio ventrale appena dietro i lobi frontali. </li></ol> Parte 4: la registrazione e la stimolazione muscolare azzurro <ol><li> Tirare un 10-20 elettrodo μω utilizzando un elettrodo estrattore elettronico (Sutter Instruments). </li><li> Riempire l&#39;elettrodo tirato con 3 M KCl. </li><li> Luogo pieno di elettrodi nel porta elettrodo e elettrodo punta manovra con un micromanipolatore vicino preparazione larvale sotto microscopio da dissezione elettrofisiologia su rig. </li><li> Identificare muscolare 6 (M6) in ogni segmento di parete del corpo (Fig. A). </li><li> Inserire con cautela elettrodo nella M6 e guardare per iperpolarizzazione rapida. Potenziali di membrana muscolare a riposo dovrebbe essere ovunque da -30 mV a -70 mV. </li><li> Regolare la luce blu in modo che la preparazione è centrata sezionato in fascio di luce. </li><li> Dare impulsi di tensione 20-100 ms per il controllo del circuito. Aumentare in misura apprezzabile tensione fornita al circuito di controllo. Guarda per i potenziali di giunzione eccitatori nella cellula muscolare (Figura 1B). </li></ol> Rappresentante dei risultati: La figura 1A mostra uno schema del sistema di registrazione e la preparazione in filetti. Figura 1B mostra EJP tipici evocato da brevi impulsi di luce. Ampiezza EJP mostrata è riassunta ampiezza da due neuroni motori conosciuta da entrambe le innervare M6. Intensità di luce inferiore solo attivato una unità di motore (dati non riportati). <img src="/files/ftp_upload/1133/figure1.png" alt="Figura 1" width="600" height="373" /> Figura 1: A) Schema generale di un impianto di registrazione intracellulare e con LED blu. Cervello (Br) viene rimosso per inibire l&#39;attività ritmica nel ganglio ventrale (VG). ChR2 è espresso in neuroni motori utilizzando il GAL4-UAS sistema. B) la registrazione intracellulare da un muscolo M6. 40 ms impulsi di luce blu (a 127 μW / mm 2) affidabile evocano grandi potenzialità sinaptiche (asterischi) in M6.

<ol>
	<li>Keshishian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545 (1996).</li><li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+development:+insights+from+Drosophila.">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35 (2007).</li><li>Lagow, R. 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=Modification+of+a+hydrophobic+layer+by+a+point+mutation+in+syntaxin+1A+regulates+the+rate+of+synaptic+vesicle+fusion.">Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.</a> PLoS Biol. 5 (4), e72 (2007).</li><li>Nagel, G., 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=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279 (2005).</li><li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940 (2003).</li><li>Schroll, 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=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr Biol. 16 (17), 1741 (2006).</li><li>Lin, D. M., Auld, V. J., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+neuronal+cell+ablation+in+the+Drosophila+embryo:+pathfinding+by+follower+growth+cones+in+the+absence+of+pioneers.">Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.</a> Neuron. 14 (4), 707 (1995).</li><li>Ralph Greenspan, . <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, 2nd ed. , (2004).</li></ol>

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Passaggi critici coinvolgono sia la dissezione iniziale e l&#39;ingresso delle cellule muscolari. Se i nervi vengono tagliati o muscolo viene danneggiato durante la dissezione iniziale è difficile continuare il resto della sperimentazione. Durante la dissezione, bisogna stare molto attenti a sezionare le forbici angolo verso l&#39;alto il più possibile durante l&#39;incisione dorsale. Durante il secondo passo fondamentale, entrando in una cellula muscolare, si deve guardare per un passato iperpolarizzazione ~ 30 mV. I...

<table><tbody><tr><td>Sylgard</td><td>Ellsworth Adhesives</td><td>4019862</td><td>&lt;a href=&quot;http://www.ellsworth.com&quot; target=&quot;_blank&quot;&gt;www.ellsworth.com&lt;/a&gt;</td></tr><tr><td>Minutens Pins</td><td>Fine Science Tools</td><td>26002-10</td><td>&lt;a href=&quot;http://www.finescience.com&quot; target=&quot;_blank&quot;&gt;www.finescience.com&lt;/a&gt;</td></tr><tr><td>Dissecting Dish</td><td>Fisher Scientific</td><td></td><td>&lt;a href=&quot;http://www.fishersci.com&quot; target=&quot;_blank&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>Neuroprobe Intracellular Amplifier and Head Stage</td><td>A-M Systems</td><td>680100</td><td>&lt;a href=&quot;http://www.a-msystems.com&quot; target=&quot;_blank&quot;&gt;www.a-msystems.com&lt;/a&gt;</td></tr><tr><td>&lt;a href=&quot;http://www.adinstruments.com/products/hardware/corporate/product/ML866/&quot; target=&quot;_blank&quot;&gt;Powerlab 4/30 data acquisition system&lt;/a&gt;</td><td><a href="http://www.adinstruments.com" target="_blank"><img src="/img/new_window.png" /> ADInstruments</a></td><td></td><td>&lt;a href=&quot;http://www.adinstruments.com&quot; target=&quot;_blank&quot;&gt;www.adinstruments.com&lt;/a&gt;</td></tr><tr><td>Grass stimulator</td><td>Grass Technologies</td><td></td><td>&lt;a href=&quot;http://www.grasstechnologies.com&quot; target=&quot;_blank&quot;&gt;www.grasstechnologies.com&lt;/a&gt;</td></tr><tr><td>Desktop Computer</td><td>Dell</td><td></td><td>&lt;a href=&quot;http://www.dell.com&quot; target=&quot;_blank&quot;&gt;www.dell.com&lt;/a&gt;</td></tr><tr><td>Dissecting Scope</td><td>Leica Microsystems</td><td></td><td>&lt;a href=&quot;http://www.leica-microsystems.com&quot; target=&quot;_blank&quot;&gt;www.leica-microsystems.com&lt;/a&gt;</td></tr><tr><td>Light Source</td><td>Dolan-Jenner Industries</td><td>41446-062</td><td>&lt;a href=&quot;http://www.dolan-jenner.com&quot; target=&quot;_blank&quot;&gt;www.dolan-jenner.com&lt;/a&gt;</td></tr><tr><td>Fly Media</td><td></td><td></td><td></td></tr><tr><td>All-Trans-Retinal</td><td>Sigma-Aldrich</td><td>116-31-4</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com&quot; target=&quot;_blank&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>&lt;em&gt;OK-371 Gal4&lt;/em&gt; Flies</td><td>Bloomington Stock center</td><td></td><td></td></tr><tr><td>&lt;em&gt;UAS-ChR2&lt;/em&gt; Flies</td><td>Fiala lab, Griffith lab</td><td></td><td></td></tr><tr><td>LED controller circuit</td><td>Built in Griffith lab</td><td></td><td>&lt;a href=&quot;http://www.ledsupply.com&quot; target=&quot;_blank&quot;&gt;http://www.ledsupply.com&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.futureelectronics.com&quot; target=&quot;_blank&quot;&gt;http://www.futureelectronics.com&lt;/a&gt;&lt;br/&gt;Composed of: &lt;br/&gt;1. 200 mA Buck Puck&lt;br/&gt;2. Blue LED&lt;br/&gt;3. Insulated wire&lt;br/&gt;4. Circuit bread board&lt;br/&gt;</td></tr><tr><td>LED Heat Sink</td><td>Thorlabs Inc.</td><td></td><td>&lt;a href=&quot;http://www.thorlabs.com/&quot; target=&quot;_blank&quot;&gt;http://www.thorlabs.com/&lt;/a&gt;</td></tr><tr><td>Air Table</td><td>TMC</td><td></td><td>&lt;a href=&quot;http://www.techmfg.com/products/accessories/intro3.html&quot; target=&quot;_blank&quot;&gt;http://www.techmfg.com/products/accessories/intro3.html&lt;/a&gt;</td></tr><tr><td>Faraday Cage</td><td>Built in Griffith lab</td><td></td><td></td></tr><tr><td>Leica Leitz M Micro-Manipulator</td><td>Leica Microsystems</td><td>ACS01</td><td>&lt;a href=&quot;http://www.leica-microsystems.com&quot; target=&quot;_blank&quot;&gt;www.leica-microsystems.com&lt;/a&gt;</td></tr><tr><td>Electrode Holder</td><td>Axon Instruments</td><td></td><td>&lt;a href=&quot;http://www.axon.com&quot; target=&quot;_blank&quot;&gt;www.axon.com&lt;/a&gt;</td></tr><tr><td>Borosilicate Glass</td><td>FHC, Inc.</td><td></td><td>&lt;a href=&quot;http://www.fh-co.com/p14-15.pdf&quot; target=&quot;_blank&quot;&gt;www.fh-co.com/p14-15.pdf&lt;/a&gt;</td></tr><tr><td>Electrode Puller</td><td>Sutter Instrument Co.</td><td></td><td>&lt;a href=&quot;http://www.sutter.com&quot; target=&quot;_blank&quot;&gt;www.sutter.com&lt;/a&gt;</td></tr><tr><td>HL 3.1 Saline with 0.8mM Ca2+</td><td></td><td></td><td>Contents (mM):&lt;br/&gt; NaCl:70 KCl:5 CaCl&lt;sub&gt;2&lt;/sub&gt;: 0.8&lt;br/&gt; MgCl&lt;sub&gt;2&lt;/sub&gt;:4Sucrose:115&lt;br/&gt;NaHCO&lt;sub&gt;3&lt;/sub&gt;: 10 Trehalose: 5 HEPES</td></tr><tr><td>Micro-Dissection Tools</td><td>Fine Science Tools</td><td></td><td>&lt;a href=&quot;http://www.finescience.com&quot; target=&quot;_blank&quot;&gt;www.finescience.com&lt;/a&gt;</td></tr></tbody></table>

channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions

Il<em&gt; Drosophila</em&gt; Larvale preparazione neuromuscolare ha dimostrato di essere uno strumento utile per studiare la fisiologia sinaptica<sup&gt; 1,2,3</sup&gt;. Attualmente, l&#39;unico mezzo a disposizione per evocare i potenziali eccitatori giunzionale (EJPs) in questa preparazione prevede l&#39;utilizzo di elettrodi di aspirazione. In entrambi i laboratori di ricerca e di insegnamento, gli studenti spesso hanno difficoltà a manovrare e manipolare questo tipo di elettrodo stimolante. Nel presente lavoro, mostriamo come stimolare a distanza i potenziali sinaptici alla larvale NMJ senza l&#39;uso di elettrodi di aspirazione. Esprimendo channelrhodopsin2 (ChR2)<sup&gt; 4,5,6</sup&gt; In<em&gt; Drosophila</em&gt; Motoneuroni utilizzando il<em&gt; GAL4-UAS</em&gt; Sistema<sup&gt; 7</sup&gt;, E fare piccole modifiche ad un impianto di base di elettrofisiologia, siamo stati in grado di evocare affidabile EJPs con impulsi di luce blu. Questa tecnica potrebbe essere particolarmente utile nei laboratori didattici neurofisiologia dove rig studente a tempo pratica e le risorse sono limitate.

Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

Questa procedura utilizza una luce blu-attivati ​​canali delle cellule algali e strumenti specifici per l&#39;espressione genetica di evocare i potenziali sinaptici con impulsi di luce a livello della giunzione neuromuscolare (NMJ) in<em&gt; Drosophila</em&gt; Larve. Questa tecnica è un poco costoso e facile da usare alternativa alla stimolazione dell&#39;elettrodo di aspirazione per gli studi di fisiologia sinaptica nella ricerca e laboratori didattici.

Channelrhodopsin2 stimolazione mediata dei potenziali Synaptic a Drosophila Giunzioni neuromuscolari

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

channelrhodopsin2-mediated-stimulation-synaptic-potentials-at

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

13.6K Views.  Brandeis. Questa procedura utilizza una luce blu-attivati ​​canali delle cellule algali e strumenti specifici per l'espressione genetica di evocare i potenziali sinaptici con impulsi di luce a livello della giunzione neuromuscolare (NMJ) in Drosophila Larve. Questa tecnica è un poco costoso e facile da usare alternativa alla stimolazione dell'elettrodo di aspirazione per gli studi di fisiologia sinaptica nella ricerca e laboratori didattici.

Video: Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila larvali NMJ Immunoistochimica

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Ricerca

Biologia

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Metodi elettrofisiologici per la registrazione Potenziali Synaptic dal NMJ Larve di Drosophila

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Optogenetic Perturbazione di attività neurale con illuminazione laser in semi-intatte<em&gt; Drosophila</em&gt; Larve in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Neuroscienze

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di<em&gt; Drosophila</em&gt; Fotorecettori e Interneuroni a luce stimoli<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da<em&gt; Drosophila</em&gt; Fotorecettori

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Macropatch focale registrazioni delle correnti sinaptiche da giunzione neuromuscolare larvale della drosofila

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac.

Esaminando monosinaptica connessioni in Drosophila utilizzando i canali di sodio tetrodotossina resistenti

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

Tintura di FM in bicicletta alla sinapsi: confronto tra alto potassio depolarizzazione, elettrica e stimolazione Channelrhodopsin

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

Mappatura del circuito a lungo raggio assistita da Channelrhodopsin dei neuroni Colliculus inferiori con Channelrhodopsins blu e rosso

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, ...

Synaptic Modulation in Drosophila After Exposure to Prolonged Light

Source: Sugie, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55176">Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light.</a> J. Vis. Exp. (120)
This video demonstrates the rearing of Drosophila melanogaster under specific light conditions to study the role of Bruchpilot proteins in neurotransmitter release and the resulting modulation of synaptic activity.

Modulazione sinaptica in Drosophila dopo esposizione a luce prolungata

1. Light Exposure Conditions

	From the larva stage until eclosion, keep the flies in a 12 h light/12 h dark cycle (LD) at 25 &#176;C (Figure 1A).
	...

Channelrhodopsin2 stimolazione mediata dei potenziali Synaptic a Drosophila Giunzioni neuromuscolari

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