eoe sub articles

EoE Sub Articles

1. Light Exposure Conditions
<ol>
	<li>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).</li>
	<li>Collect the flies 0 - 6 h after eclosion and place them into a new vial containing fly food.</li>
	<li>Set the vials into a transparent acrylic resin rack with three steps (a height of 41 cm, a base of 21 cm, a thickness of 4 cm, and a height of 13 cm for each step) (Figure 1B).</li>
	<li>Adjust the distance between the rack and an LED panel to provide illumination with an average intensity of 1,000 lux using a digital light meter (Figure 1C).</li>
	<li>Keep the flies for 1 - 3 d under one of the following conditions: constant darkness (DD), 12 h light/12 h dark cycle (LD), or constant light (LL) at 25 &#176;C in a small incubator (Figures 1A and 1C).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vial</td>
			<td>Hightech, Japan</td>
			<td>MKC-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Plug</td>
			<td>Thermo Fisher Sciehtific, USA</td>
			<td>AS-275</td>
			<td></td>
		</tr>
		<tr>
			<td>Customized transparent rack made of acrylic resin</td>
			<td>Shin-Shin Corporation, Japan</td>
			<td></td>
			<td>a height of 41 cm, a base of 21 cm, a thickness of 4 cm and a height of 13 cm for each step</td>
		</tr>
		<tr>
			<td>Cool incubator</td>
			<td>MITSUBISHI ELECTRIC, Japan</td>
			<td>CN-40A</td>
			<td></td>
		</tr>
		<tr>
			<td>LED panel</td>
			<td>MISUMI, Japan</td>
			<td>LEDXC170-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital light meter</td>
			<td>CEM</td>
			<td>DT-1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly pad</td>
			<td>Tokken, Japan</td>
			<td>TK-HA03-S</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/22855/22855_Figure1.jpg" /> 
Figure 1: Preparation of Conditions for Exposure to Defined Light Intensity. (A) Light exposure protocols after eclosion. DD, continuous darkness; LD, 12 h light/12 h dark; LL, constant exposure to light. Fly brains were dissected at the times indicated in the brackets (Adapted from a previous publication9). (B) Vials are aligned in a customized acrylic transparent rack. (C) The acrylic rack and ...

Synaptic Modulation in Drosophila After Exposure to Prolonged Light

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).
	...

Begin with freshly eclosed Drosophila melanogaster flies in a collection vial containing nutrients.
Place these vials in a transparent acrylic rack.
Position the rack inside an incubator equipped with an internal light source at a precise distance to provide uniform and desired light exposure.
Rear the flies under these light conditions for a prolonged period.
In Drosophila eyes, the photoreceptor cells act as sensory neurons, detecting the light and converting it into electrical signals.
These signals travel along the axon and reach the synaptic terminals, where they connect with other neurons.
These terminals possess the Bruchpilot proteins, which facilitate the fusion of neurotransmitter-carrying vesicles to the cell membrane, allowing neurotransmitter release.
The released neurotransmitters bind to the receptors on the postsynaptic neurons to transmit the electrical signal, leading to synaptic activity.
 With continuous exposure to light, the Bruchpilot protein level decreases, reducing neurotransmitter release and resulting in the plasticity or modulation of synaptic activity.

synaptic-modulation-in-drosophila-after-exposure-to-prolonged-light

Universidad de Cartagena UDEC

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Neurophysiology

Stimulation and Modulation Techniques

63 Views. Fonte: Sugie, A., et al. Analisi della modulazione sinaptica dei fotorecettori di Drosophila melanogaster dopo l'esposizione alla luce prolungata. J. Vis. Exp. E 120 Questo video dimostra l'allevamento di Drosophila melanogaster in specifiche condizioni di luce per studiare il ruolo delle proteine Bruchpilot nel rilascio di neurotrasmettitori e la conseguente modulazione dell'attività sinaptica. 

Video: Modulazione sinaptica in Drosophila dopo esposizione a luce prolungata - Concept

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 ...

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JoVE Journal

Neuroscienze

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, ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Metodi elettrofisiologici per misurare i livelli di fotopigmento nei fotorecettori di Drosophila

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

Synaptic Modulation in Drosophila After Exposure to Prolonged Light

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Synaptic Modulation in Drosophila After Exposure to Prolonged Light