Drosophila Adult Brain Dissection: A Method in Fly Neurobiology

Abstract

Source: Kelly, S. M., et al. Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains. J. Vis. Exp. (2017).

This video describes how to dissect and isolate the adult Drosophila brain–a procedure necessary for visualizing the organ, which is otherwise obscured by the cuticle, eye, and tracheal tissue. The example protocol shows a detailed demonstration yielding high-quality preparations that can be used for immunostaining and imaging methods in Drosophila neurobiology.

Protocol

This protocol is an excerpt from Kelly et al., Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains, J. Vis. Exp. (2017).

1. Dissection Station Preparation

1. Position the stereomicroscope and light source with attached fiber optic goosenecks on a large benchtop. To promote steady hand movements and reduce hand "shake" while dissecting, it is essential that adequate hand and arm rest space is available around the microscope. Ensure that there is approximately 8 – 10 inches on either side of the microscope and 4 – 6 inches between the base of microscope and edge of the bench.
2. Fill 2 or 3 wells of a glass 9-well or 3-well dish with 1.0 mL of PTN buffer (0.1 M Sodium Phosphate Buffer pH 7.2, 0.1% nonionic surfactant, see Table of Materials for complete buffer components) and place next to the dissecting station on ice. Newly dissected brains will be transferred to this dish and stored until the fixation step.
   NOTE: If live imaging is required, dissection buffer should be 1x Phosphate Buffered Saline (PBS) or HL3 buffer. If intracellular protein localization is required, PBS can also be used as an alternative buffer for dissection and fixation. Following fixation, permeabilization of cell membranes should then be performed using PTN washes containing 0.1% or 0.3% nonionic detergent.
   1. If PBS is used for dissections and fixation, pipette tips should be rinsed at least one time with a detergent-containing buffer (such as PTN) to prevent brains from sticking to the plastic pipette tips during transfer to microcentrifuge tubes.
3. Using an empty 35 mm glass or plastic petri dish, construct a dissection dish containing a silicone elastomer. Briefly, mix the elastomer components according to the manufacturer's directions, pour into 35 mm dishes, and let it polymerize overnight on a flat surface. Elastomer containing dissection dishes should be used to protect the fine tips of dissecting forceps, which can easily be damaged if contact is made between the forceps and a harder surface, such as a glass dish. We also regularly purchase commercially available silicone coated dishes from online retailers. To increase contrast during dissections, silicone elastomer dissection dishes containing inactivated charcoal (and thus colored black) are particularly useful.

2. Adult Brain Dissection Procedure

1. Anesthetize 3 – 5 day old adult D. melanogaster with CO₂ or by using ice. If using ice, place the vial containing flies upside down (plug end down) into an ice bucket for ~5 min. Placing the vial into ice upside down prevents flies from becoming lodged in the food. Once flies have been anesthetized, place the flies on a cold metal pad or petri dish sitting in ice or on a CO₂ emitting fly pad. If dissecting brains to analyze neurodegeneration, older flies may also be used.
2. Place a small amount (150 – 200 µL) of PTN in the center of the dissection dish using a transfer pipette or a p200 pipette to create a "bubble" of PTN. Place the dissection dish under the stereomicroscope and adjust the lighting and focus so that the bubble of PTN fills the field of view and is uniformly illuminated.
3. Manipulate flies so that they are "belly up" (i.e., ventral side up) while lying on the metal or CO₂ pad.
4. Using one pair of #5 forceps, grasp the abdomen of a fly to be dissected and, keeping hold of the fly, completely submerge it in the PTN on the dissection dish.
   NOTE: For the remainder of the protocol, all steps should be performed while the head is submerged in PTN.
5. Using a second pair of #5 forceps, grasp the base of the fly proboscis and pull the two pairs of forceps apart to detach the fly head from the body. Discard the abdomen and thorax. During this step, it is critical that the head is not released and allowed to float on the surface of the PTN. Once the head is floating, it can be very difficult to grasp again without crushing the brain.
   NOTE: Using this method, connections between the brain and ventral nerve cord are severed. If intact connections between these regions of the CNS are required, an alternative dissection protocol should be followed. If the proboscis detaches from the fly head before the head is removed, there will be a hole where the proboscis was. In this case, grasp the fly head at the edge of the hole near one eye. Then remove the head using a moderate amount of force while pulling the two pairs of forceps apart from one another. Occasionally, when the head is removed from the body, the gut and/or ventral nerve cord remains attached to the head and should be removed before continuing the dissection.
6. While one pair of forceps grasps the proboscis, the second pair should grasp the medial edge of the right fly eye. Slowly, pull the forceps apart from one another. This step should be performed with a small amount of steady lateral force. As the forceps slowly move apart from one another, the proboscis should pull away from the head and create a central hole in the head cuticle. Discard the proboscis with the first pair of forceps without releasing the medial portion of the right eye from the second pair.
   NOTE: The adult D. melanogaster brain is in the caudal (i.e., posterior/rear) region of the fly head. Thus, grasping that region of the head should be avoided. Ideally, only the rostral (i.e., front) portion of the head near the medial retina should be directly grasped by the forceps. The brain and associated trachea should now be visible through the central hole in the cuticle. At this time, any white stringy threads of trachea protruding from the hole can be removed and discarded.
7. With the second pair of forceps, grasp the medial edge of the left retina (at the edge of the central hole in the head cuticle). To remove the retinas and associated cuticle, slowly pull the forceps away from one another at a 180° angle. As the retina dissociates from the underlying optic lobe, you should feel a slight decrease in tension. Proceed slowly to prevent tearing the optic lobe.
   NOTE: Separating the forceps too quickly during this step may result in the tearing of the optic lobe or disruption of mushroom body structures. Occasionally, the cuticle will be removed but pieces of the retina will remain attached to the optic lobe. If imaging the mushroom bodies, it is not completely necessary to remove the entire retina. However, analysis of other brain regions (such as retinal neuron innervation of the optic brain lobes) may require the retina to be completely removed as described by.
   NOTE: As the retina is slowly separated from the underlying optic lobe of the brain, the optic lobe should be observable as an opaque white structure covered with white, stringy trachea. Once one retina has been removed, it can be discarded. When analyzing pathfinding of retinal neurons, particular care should be taken during this step to prevent damage to the optic lobe. An additional protocol focused on dissection and live imaging of the photoreceptor neurons is also available.
8. Now, carefully remove as much of the visible trachea as possible. The trachea may already contain or later fill with air, causing brains to float and, potentially, be lost during later immunostaining steps. To remove trachea, pick it off the brain using a very sharp pair of #5 forceps.
9. Remove the remaining retina and surrounding cuticle by using both pairs of forceps to grasp the medial region of the left fly retina. Carefully tear the retina in half to remove pieces of the retina and cuticle. In some cases, removing the remaining cuticle without crushing the brain proves especially challenging. In these cases, we have found that the remaining strands of the ventral nerve cord can instead be grasped by one pair of forceps while the other pair of forceps is used to carefully remove the last of the cuticle.
10. Using a p200 pipette, move dissected brains to one well of the 9- or 3-well dish containing PTN. Brains of the same genotype should be pooled together into the same well and kept on ice. Brain tissue should be fixed within one hour of dissection. Brains can be fixed in small batches and pooled if a larger number of brains is required. In most circumstances, an experienced researcher can usually dissect a brain and transfer it to the glass collection dish in approximately 3 – 5 min.

Materials

Microdissection forceps/tweezers	Ted Pella	505-NM
Sylgard dishes	Living Systems Instrumentation	DD-50-S-BLK	Available from amazon.com
Na Phosphate Buffer monobasic	Sigma	S3139
Triton X 100	Sigma	X100-100ml
stereomicroscope	Leica S6D with KL300 LED light source
9-well dish (spot plate)	VWR	89090-482
35mm dish	Genesee Scientific	32-103
Sylgard	Fisher	50-366-794
Kimwipe	Fisher	06-666
PTN Buffer			0.1M NaPhosphate, pH 7.2, 0.1% Triton-X-100, Typically make up 0.5 L of 0.1M NaPhosphate buffer and aliquote 50ml at a time as needed