inducing penetrating traumatic brain injury in adult drosophila flies

Inducing Penetrating Traumatic Brain Injury in Adult Drosophila Flies

After anesthetizing the flies on a carbon dioxide pad, sort newly eclosed F1 young male flies within six hours of eclosion, and place them in clean vials containing food with 40 or fewer flies per vial. Next, sanitize minutien pins by placing approximately 100 pins in a 1.5-milliliter microcentrifuge tube filled with 70% ethanol for five minutes. Then, sanitize the carbon dioxide pad in paintbrush by spraying 70% ethanol and wiping dry with a clean, lint-free tissue.
Once the tools are clean and dry, transfer 40 or fewer sorted F1 males onto the clean pad and separate them into two groups. One group will serve as the control uninjured flies, and the second experimental group will be subjected to penetrating traumatic brain injury. Next, using forceps, pull four to five new minutien pins out of the microcentrifuge tube, and place them near the edge of the carbon dioxide pad. Then, under the dissecting scope, choose a straight minutien pin with a sharp point. Reuse sharp pins and place damaged or blunt pins in a separate tube containing 70% ethanol for safe disposal.
Next, for flies with the standard cross genotype, switch on the stereomicroscope LED lamp equipped with the appropriate excitation and emission filters for the green fluorescent protein, which permits excitation at 440 to 460 nanometers and allows visualization at 500 to 560 nanometers. Then, choose a fly from the experimental group, and position the fly such that the experimenter has a dorsal view of the head capsule with the fly&#39;s head to the right. Using forceps, pick up and hold the selected minutien pin in one hand, and the paintbrush in the other hand. Then, place the brush on the interior of the dorsal thorax, and push down gently to stabilize the fly.
Aim the minutien pin&#39;s tip at the mushroom bodies, cell bodies on the right side of the head, and penetrate the head capsule. If using landmarks, target the dorsal head cuticle between the ocelli and the dorsal rim of the eye. After completing the injury, use the paintbrush to push the head gently off the minutien pin. If using the brain for RNA sequencing or qRT-PCR, make a second injury on the left side of the head. Once all experimental flies have been injured, place the control and injured flies into separate labeled vials containing food.

inducing-penetrating-traumatic-brain-injury-in-adult-drosophila-flies

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1. Penetrating traumatic brain injury (PTBI;&#160; Figure 1)
<ol>
	<li>Sort newly eclosed F1 flies. Select young males within 6 h post-eclosion. Place these males in clean vials containing food, with 40 or fewer flies per vial. 
	&#160;NOTE: This is most easily accomplished in mid-morning by anesthetizing flies on the CO2 pad and identifying adult males that still have meconium (visible as a dark greenish spot through the abdominal wall) in their guts.</li>
	<li>Sanitize Minutien pins (see Table of Materials) for at least 5 min by placing ~100 pins in a 1.5 mL microcentrifuge tube filled with 70% ethanol.</li>
	<li>Sanitize the CO2 pad and a paintbrush by spraying 70% ethanol and wipe dry with a clean, lint-free tissue. Once the tools are clean and dry, transfer 40 or fewer sorted F1 males back onto the clean pad.</li>
	<li>Separate the F1 males into 2 groups on the fly pad. One group will serve as a control, uninjured flies. The second experimental group will be subjected to PTBI.</li>
	<li>Using forceps, pull 4-5 new Minutien pins out of the microcentrifuge tube and place them near the edge of the CO2 pad. Under the dissecting scope, choose a straight Minutien pin with a sharp point. 
	&#160;NOTE: Using a single Minutien pin for an experiment will reduce variability. Reuse sharp pins. Place damaged or blunt pins in a separate 1.5 mL microcentrifuge tube containing 70% ethanol for safe disposal.</li>
	<li>If working with flies from the standard cross, switch on the stereo microscope light-emitting diode (LED) lamp equipped with the appropriate excitation and emission filters for green fluorescence protein (GFP). This permits excitation at 440-460 nM and allows visualization of 500-560 nM. 
	&#160;NOTE: With this lamp and filter set, the cell bodies of the mushroom body will fluoresce green through the head cuticle (Figure 1C). For perma-twin flies or flies of other genotypes, a standard white light illuminator for the stereo microscope can be used to visualize landmarks on the head cuticle for targeting the injury to the mushroom body (Figure 1C).</li>
	<li>Use the forceps to pick up and hold the selected Minutien pin in one hand (for right-handed people, this is usually the right hand) and the paintbrush in the other (usually left) hand. Choose a fly from the experimental group and position the fly such that you have a dorsal view of the head capsule with the fly&#39;s head to the right. Place the brush on the anterior of the dorsal thorax and push down gently to stabilize the fly.</li>
	<li>Aim the Minutien pin&#39;s tip at the mushroom body&#39;s cell bodies on the right side of the head and penetrate the head capsule. If using landmarks, target the dorsal head cuticle between the ocelli and the dorsal rim of the eye (Figure 1).</li>
	<li>After completing the injury, use the paintbrush to push the head off the Minutien pin gently.</li>
	<li>If using the brain for RNA-Seq or qRT-PCR, make a second injury on the left side of the head.</li>
	<li>Repeat steps 2.8-2.10 to injure all of the flies in the experimental group.</li>
	<li>Once all flies have been injured, place the control and injured flies into labeled, separate vials containing food. Lay the vials horizontally (i.e., on their sides) while the flies recover from the anesthesia and during subsequent aging to prevent flies from getting caught in the food.</li>
	<li>Place standard cross flies at 25 &#176;C, and perma-twin flies at 30 &#176;C to age.</li>
	<li>For flies aged longer than 24 h, place them on clean food every 1-2 days.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carbon dioxide bubbler</td>
			<td>Genesee Scientific</td>
			<td>59-181</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Carbon dioxide pad</td>
			<td>Genesee Scientific</td>
			<td>59-114</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>cotton plugs</td>
			<td>Genesee Scientific</td>
			<td>51-101</td>
			<td>For EdU labeling</td>
		</tr>
		<tr>
			<td>Drosophila vials</td>
			<td>Genesee Scientific</td>
			<td>32-109</td>
			<td>For EdU labeling</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>252549</td>
			<td>For fixing adult brains, added to PEM</td>
		</tr>
		<tr>
			<td>Minutien pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>For brain injury; 12.5 &#956;m diameter tip and 100 &#956;m diameter rod</td>
		</tr>
		<tr>
			<td>NIGHTSEA stereo microscope fluorescence adaptor</td>
			<td>Electron Microscopy Sciences</td>
			<td>SFA-GR</td>
			<td>Fluorescence setup for dissecting microscope</td>
		</tr>
		<tr>
			<td>phosphate buffered saliine (PBS)</td>
			<td>components sourced from various companies</td>
			<td></td>
			<td>For dissecting brains and making immunohistochemistry blocking and washing solutions; 100 mM of K2HPO4, 140 mM of NaCl, pH 7.0</td>
		</tr>
		<tr>
			<td>phosphate buffered saline with 0.1% Triton X-100 (PT)</td>
			<td>components sourced from various companies</td>
			<td></td>
			<td>For washing dissected brains</td>
		</tr>
		<tr>
			<td>Two pair of #5 watchmakers forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td>Used to hold the Minutien pins and for brain dissections</td>
		</tr>
	</tbody>
</table>

Source: Crocker, K. L., et al. <a href="https://app.jove.com/v/63182">Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain.</a> J. Vis. Exp. (2021)
This video demonstrates the induction of penetrating traumatic brain injury in the drosophila brain. Newly emerged flies are selected, anesthetized, and positioned under a fluorescence-equipped stereo microscope. The GFP-expressing mushroom body is identified, and a Minutien pin is inserted to cause injury. The flies are allowed to recover for further studies.

<img alt="Figure 1" src="/files/ftp_upload/23200/23200_Figure1.jpg" /> 
Figure 1: Penetrating Traumatic Brain Injury (PTBI) to the adult Drosophila central brain. (A) Schematic of the exterior of an adult fly head. This is a frontal view. Thus, the right side of the animal is to the viewer&#39;s left. (B) Schematic of the interior of an adult Drosophila head with the injury trajectory indicated in grey. This is a posterior view. Thus, in this image and subsequent figures, the right side of the brain is to the right. Central brain PTBI impacts multiple brain structures, including the mushroom body (green) and tissues outside the brain, including the fat body (blue) and hemocytes (red). CB = central brain region. OL= optic lobe region. (C) Dorsal view of a live adult head in which mushroom bodies (arrowheads) are labeled with a green fluorescent protein (GFP). This is the &#39;standard genotype&#39; (see text for details). The PTBI protocol reproducibly results in injury to the mushroom bodies.

Source: Crocker, K. L., et al. Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain. J. Vis. Exp. (2021)
This video demonstrates ...

Take newly emerged, genetically modified adult Drosophila flies.&#160;
Mushroom bodies in the fly&#39;s brain contain neurons that express green fluorescent proteins.
Mushroom bodies are structures containing complex neuronal networks essential for learning and memory.
Anesthetize the flies on a carbon dioxide pad.
Under a stereo microscope equipped with the required filters, visualize the mushroom bodies expressing green fluorescence.
Take a sanitized Minutien pin and push it through the head capsule into the mushroom body.
This causes damage to mushroom body neurons and disrupts neuronal networks, inflicting penetrating traumatic brain injury.
Next, gently remove the pin from the fly.
Place the injured flies in a vial with food and allow them to recover for further studies.

6 Views. Inducing Penetrating Traumatic Brain Injury in Adult Drosophila Flies

Video: Inducing Penetrating Traumatic Brain Injury in Adult Drosophila Flies - Experiment

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Inducing Penetrating Traumatic Brain Injury in Adult Drosophila Flies

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Inducing Penetrating Traumatic Brain Injury in Adult Drosophila Flies