dissection and mounting of fruit fly larval brain explants

Dissection and Mounting of Fruit Fly Larval Brain Explants

To harvest brains from larval Drosophila, under a dissecting microscope, use one pair of number 5 standard-tip dissection forceps to hold a larval specimen in place, while using the other to carefully dissect out the brain, preserving the eye disks, brain lobes, and ventral nerve cord. Then, remove the attached muscles to minimize any movement of the sample during imaging. When all of the brains have been harvested, use vacuum grease to draw a square chamber onto a glass slide, and add 20 microliters of external saline solution to the chamber.
Use the forceps to transfer the brains to the chamber and adjust the positions of the brains under the microscope, with the dorsal sides facing up. Cover the chamber with a glass coverslip and press gently on the coverslip to reduce sample drifting during the imaging procedure.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dissecting and Mounting Larval Brain Explants
<ol>
	<li>Dissect larval brains in the physiological external saline solution (120 mM NaCl, 4 mM MgCl2, 3 mM KCl, 10 mM NaHCO3, 10 mM Glucose, 10 mM Sucrose, 5 mM N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mM Ca2+, pH 7.2) under a dissection microscope (4.5x magnification power) with two pairs of #5 standard tip dissection forceps (11 cm). Use one pair of forceps to hold the larval body in place and the other to carefully dissect out the brain. Preserve the eye disks, brain lobes and the ventral nerve cord. Remove attached muscles to minimize sample movements during imaging.</li>
	<li>Prepare a glass slide (25 x 75 x 1.0 mm3) and use a syringe to draw a square chamber with vacuum grease.</li>
	<li>Add 20 &#181;L of external saline solution to the square chamber with grease barriers.</li>
	<li>Transfer dissected larval brains into the chamber on the glass slide using forceps. Adjust the position of the brains under the dissection scope to ensure the dorsal side faces up.</li>
	<li>Cover the chamber with a glass cover slip (22 x 22 x 0.15 mm3). The larval brain is now mounted on the slide within a chamber filled with the external saline solution (Figure 1B). 
	NOTE: Pressing gently on the coverslip confines the brains and reduces sample drifting in the subsequent imaging session.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>high vacuum grease</td>
			<td>Dow Corning</td>
			<td>79751-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Sheng, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60287">Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons.</a> J. Vis. Exp. (2019).
This video demonstrates the dissection and mounting of brain explants from Drosophila larvae. The central nervous system (CNS), attached to eye disks, is extracted from the larvae and then mounted on a glass slide in a physiological saline solution.

<img alt="Figure 1" src="/files/ftp_upload/22634/22634_Figure 1.jpg" /> 
Figure 1: Workflow of the dendrite dynamics imaging and quantification protocol. (A) The protocol contains six steps covering sample preparation, image collection, image processing and semi-automated quantification of dendrite dynamics. (B) A schematic diagram illustrating an imaging chamber containing a larval brain explant mounted with the dorsal side up. The brain explant has an individually labeled ventral lateral neuron (LNv) in each lobe and is immersed in the external saline solution. The vacuum grease barriers support the weight of the coverslip and forms a small chamber that prevents the brain from moving.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with the larvae of Drosophila, a fruit fly, in a physiological saline solution inside a dish. The solution prevents the larvae from dehydrating.
Under a dissection microscope, locate the anterior end of the larvae with the mouth hooks.
The anterior end contains the eye disks and the central nervous system, or CNS, which comprises the brain lobes and a ventral nerve cord.
Hold the posterior end of the larva using forceps. With another pair of forceps, pull the anterior end to extract the internal organs.
Remove the gut and attached muscles to isolate the CNS attached to the eye disks.
To mount the brain explant, transfer the extracted tissue onto the saline solution enclosed within a grease barrier on a glass slide.
Ensure that the dorsal side of the explant is facing up, and seal the tissue with a coverslip. The brain explant is ready for analysis.

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视频: 果蝇幼虫脑外植体的解剖和镶嵌 - 实验

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

科研

JoVE Journal

发育生物学

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

神经科学

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

光斑分析分析果蝇拉瓦尔光轴

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Dissection and Mounting of Fruit Fly Larval Brain Explants

成績單

Dissection and Mounting of Fruit Fly Larval Brain Explants