eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Dissect and Prepare the Larval Drosophila CNS
NOTE: Methods for larval CNS dissection are clearly illustrated in Hafer and Schedl, but these previously published methods reduce the length of the descending neurons that are important for measuring spike frequency. Here, an additional method is outlined to excise the larval CNS that maintains long, intact descending neurons.
<ol>
	<li>Saline preparation.
	<ol>
		<li>Prepare the dissection and recording saline to the following in mM: 157 NaCl, 3 KCl, 2 CaCl2, 4 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES); titrate pH to 7.25.</li>
	</ol>
	</li>
	<li>Dissect the larval Drosophila CNS.
	<ol>
		<li>Identify and extract a wandering third-instar Drosophila melanogaster from the culture vial and place into 200 &#181;L of saline (Figure 1A).</li>
		<li>Grasp the mouth hooks with a pair of fine forceps and grasp the abdomen of the maggot with a second pair of forceps (Figure 1B). 
		NOTE: Do not apply too much pressure to the abdomen as this can result in tearing of the thin cuticular layer of the maggot.</li>
		<li>Gently pull the mouth hooks and abdomen in different directions to separate the caudal end of the maggot from the head region and expose the viscera of the maggot (Figure 1C). 
		NOTE: The CNS will be intertwined with the trachea and digestive tract. Do not cut the CNS away from any tissue to prevent cutting the descending peripheral nerves.</li>
		<li>Tease the CNS out of the digestive tract and trachea with forceps (Figure 1D).</li>
		<li>If necessary, disrupt the blood brain barrier by manually transecting the CNS posterior to the cerebral lobes with Vannas spring scissors. 
		NOTE: This should be done based on the physiochemical properties of the chemical being used. The red line in Figure 2A is the suggested transection point and the transected CNS with intact descending peripheral nerve trunks shown in Figure 2B. The transected CNS is ready for experimentation after completion of step 1.2.5.</li>
	</ol>
	</li>
</ol>
2. Extracellular Recording of Drosophila CNS.
<ol>
	<li>Prepare the CNS for recording.
	<ol>
		<li>Pull a glass pipette electrode from borosilicate glass capillaries to a resistance of 5-15 m&#937;.</li>
		<li>Insert the transected CNS into the wax chamber that contains 200 &#181;L of saline. Clamp an uncoated insect pin with the alligator clip soldered to the ground wire and insert the pin into the saline to complete the circuit.</li>
		<li>Using the micromanipulators, orient the electrode to the caudal end of the transected CNS. Eliminate recording of background noise by adjusting the threshold level in the acquisition/analysis software prior to contacting the peripheral nerve trunks.</li>
		<li>Draw any convenient peripheral nerves into the suction electrode by applying slight negative pressure on the syringe. 
		NOTE: The baseline firing frequency is correlated to the number of neurons drawn into the electrode where more neurons oftentimes result in increased resistance of the electrode.</li>
	</ol>
	</li>
	<li>Begin extracellular recording of CNS descending nerve activity.
	<ol>
		<li>Start the recording on the data acquisition software and monitor the baseline firing frequency. Allow the firing rate to equilibrate for 5 min prior to collecting baseline firing rate data.</li>
		<li>Discard the preparation and recording if the pattern of firing of control treatment is not a bursting pattern similar to that shown in Figure 3A. 
		NOTE: Altered pattern of firing suggests abnormal or unstable activity of the central pattern generator, which can alter neural function.</li>
		<li>After 5 min, add 200 &#181;L of saline + vehicle to bring the total volume of the chamber to 400 &#181;L to begin recording control firing rates.</li>
		<li>After baseline has been established (3-5 min), withdraw 200 &#181;L of saline and add 200 &#181;L of the experimental agent solubilized in saline. 
		NOTE: Dimethyl sulfoxide (DMSO) is the recommended solvent for lipophilic compounds and should not exceed 0.1% v/v.</li>
		<li>Apply drug concentrations in a serial manner (low to high concentrations) and record each concentration for a select period of time (determined by the investigator based on the chemical properties of the drug). Label this time point of drug application in the acquisition/analysis software by including a comment that includes the drug and the final concentration. 
		NOTE: Firing activity of the CNS will decline by approximately 10-20% after 30-50 min after the dissection and therefore, appropriate untreated controls should be performed, and drug treatments should not exceed this time period.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Drosophila melanogaster&#160;(strain OR)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>2376</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Kinetic Systems</td>
			<td>9200 series</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Kinetic Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope on a Boom</td>
			<td>Nikon</td>
			<td>SMZ800N</td>
			<td>Multiple scopes can be used; boom stand is critical</td>
		</tr>
		<tr>
			<td>AC/DC differential amplifier</td>
			<td>ADInstruments</td>
			<td>AM3000H</td>
			<td>The model 1700 can be used instead of the model 3000</td>
		</tr>
		<tr>
			<td>audio monitor</td>
			<td>ADInstruments</td>
			<td>AM3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Hum Bug Noise Eliminator</td>
			<td>A-M Systems</td>
			<td>726300</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System (PowerLab)</td>
			<td>ADInstruments</td>
			<td>PL3504</td>
			<td>Multiple PowerLab models can be used.</td>
		</tr>
		<tr>
			<td>Lab Chart Pro Software</td>
			<td>ADInstruments</td>
			<td>N/A - Online Download</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Lights</td>
			<td>Edmund Optics</td>
			<td>89-740</td>
			<td>Different light sources can be used, but fiber optics are the most adaptable</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M325</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Holder</td>
			<td>World Precision Instruments</td>
			<td>MEH715</td>
			<td>Different models are acceptable</td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td>World Precision Instruments</td>
			<td>multiple based on size</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>PG52151-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>Also can use Narashige PC-100</td>
		</tr>
		<tr>
			<td>Black Wax</td>
			<td>Carolina Biological Supply</td>
			<td>974228</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-coated insect pins, size #2</td>
			<td>Bioquip</td>
			<td>1208S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fince Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
	</tbody>
</table>

extracellular recording of electrical activity in the drosophila central nervous system

Source: Swale, D. R., et al. <a href="https://app.jove.com/v/58375">Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster.</a> J. Vis. Exp. (2018)
The video demonstrates the extracellular recording of neuronal activity from the central nervous system (CNS) of Drosophila larvae. The transected CNS is positioned in a recording chamber, and the electrical activity from a descending nerve is recorded to establish the baseline firing frequency. A test agent is then introduced, and the change in firing frequency is recorded to determine the agent&#39;s excitatory or inhibitory effect.

<img alt="Figure 1" src="/files/ftp_upload/22761/22761_Figure1.jpg" /> 
Figure 1: Method for excising the CNS from third-instar maggots. (A)&#160;Intact maggot submerged in 200 &#181;L of saline. The arrow indicates the mouth hooks that are used for separation of the body wall.&#160;(B)&#160;Two pairs of forceps are placed in the middle of the maggot and on the mouth hooks to begin separation of the body wall.&#160;(C)&#160;Body wall...

Extracellular Recording of Electrical Activity in the Drosophila Central Nervous System

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Dissect and Prepare the Larval ...

Place a transected central nervous system, or CNS, of a Drosophila larva inside a recording chamber filled with saline solution.
The transected CNS includes the ventral ganglia and descending peripheral nerves.
Place a suction electrode at the posterior end of the transected CNS. Connect the chamber to a grounding wire to reduce background noise.
Apply negative pressure to draw a peripheral nerve into the electrode and initiate extracellular recording of the nerve activity.
Monitor the spontaneous generation of action potentials by neurons over time, termed the baseline firing frequency, and allow it to stabilize.
Add a solvent as a control. Record the activity to ensure the firing frequency remains consistent with the baseline.
Introduce the target test agent dissolved in the solvent and record the firing frequency.
An increase in frequency exhibits the agent&#39;s excitatory effect, whereas a decrease in frequency indicates an inhibitory effect.

extracellular-recording-electrical-activity-drosophila-central

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

67 조회수. 출처: Swale, D. R., et al. Third-Instar Drosophila Melanogaster의 중추 신경계 활동의 전기 생리학적 기록. J. Vis. 특급 (2018) 이 비디오는 초파리 유충의 중추신경계(CNS)에서 신경 세포 활동을 세포 외로 기록하는 것을 보여줍니다. transection된 CNS는 기록 챔버에 배치되고 하행 신경의 전기 활동을 기록하여 기준선 발사 빈도를 설정합니다. 그런 다음 테스트 에이전트를 도입하고 발사 빈도의 ?...

비디오: 초파리 중추신경계의 전기적 활동에 대한 세포외 기록 - 개념

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Zebrafish 초기 배아에 종양 세포 전기 활동의 시각화

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

연구

JoVE Journal

발생학

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

에서 전기 생리 학적 기록<em&gt; Drosophila</em&gt; 낮은 휘발성의 냄새에 반응하는 Trichoid Sensilla

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

신경과학

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

단일 셀 해상도 형광 애벌레 뇌 문화에 살고 있는 초파리 Circadian Clock의 이미징

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Extracellular Recording of Electrical Activity in the Drosophila Central Nervous System

내레이션 대본

Extracellular Recording of Electrical Activity in the Drosophila Central Nervous System