Dissection of Drosophila CNSs
ITO Kei: 1989 May; revised 1995 June
English Version 1999 June

A: Tools
I: Forceps
A pair of INOX No.5
Three types of material are available : Stainless, Taxal and Titanium (from Fontax Inc. Switzerland)
Among them Taxal is the best. Titanium, which is the most expensive, is somewhat too soft.
Sharpen the tip of the forceps:
Method A: sharpen with Arkansas stone (fine oil stone)
Can be obtained as the sharpener for drawing pen. A milk-white, translucent one is better than ablack one.
Sharpen the forceps under a dissecting microscope.
Method B: sharpen with sandpaper. (use #1000 to shape the tip, #2000 for finishing.)
Just after purchaseThick tips

Thick-tipThin-tip type (2 pairs needed)
One thick, the other sharp

Thin (sharp) tips

One thick, the other sharp

to grasp larvae and pupae
for CNS dissection
to peel the pupal cuticle without damaging the inside

II: microscalpel (optional)
Sharpen the tip of a sewing needle
Recommended product : #A102.9, No. 20 needle made by Milward Needles Co. (UnitedKingdom)
A: Put the needle to a needle holder
B: Sharpen the tip under a dissecting microscope. First, keep the holder upright and cut the tip ofthe needle to make it blunt. Then, shape two sides of the needle to make it like a small knife.

- 1 -B: Dissection of the CNS
The way of dissecting the central nervous system (brain and the thoracic ganglia) depends on thedevelopmental stages.
Dissection is performed in a small dish filled with PBS. Put sheets of tissue paper on both sides of thedish. Transfer the remnants of dissected bodies (legs and wings, thorax and abdomen, head capsule,etc.) to this paper and clean the forceps. Otherwise the PBS may become full of debris.
After Dissection, transfer the specimen to an Eppendorf tube filled with a small amount (ca. 200 µl) of
fixative with a 200-µl pipette. Pool the specimen for up to 20 minutes. (i.e. 5 to 20 CNSs depending on
how fast you can dissect.) in case of Formardehyde fixation, which should last for 50-70 min. (Poolfor a shorter time for the fixation with shorter incubation period.) Each time you transfer the specimen,
the fixative is diluted with the PBS you bring with the specimen. After pooling all the specimen, remove
the diluted fixative and add enough amount of fresh fixative.
Speed is more important than perfection. After the head cuticle is removed, transfer the specimen to thefixative as soon as possible. Discs, tracheae and fat bodies can be removed after fixation. (Note: Airsacs of the adult brains are better removed, since the air inside the sacs may put the specimen afloat in
the staining solution.)- 2 -I: Dissecting larval CNSs
Essentially the same as the dissection of salivary grand.
Compare your forceps. One pair of them may have thicker tips than the others. Thicker tips areconvenient for grasping the animal without damaging their body. Sharper ones are convenient forremoving small tissues.
1: Grasp the mouth part of a larva with the thicker forceps.
2: Grasp the “neck” (about one fourth from the head) of the animal with the sharper forceps, and pull
the body longitudinally.
3: Neck cuticle is torn apart. Internal organs, i.e. the oesophagus, gut, brain and salivary grand will spillout.
4: Locate the CNS. Remove other tissue.

II: Dissecting early pupae (before pupation: 0-12 hours APF)
In diptrans such as Drosophila, the cuticle of larvae remains after the final molt to become thepuparium. The hardening of the larval cuticle is called “puparium formation”. After 10-15 hours, thepupal cuticle is formed beneath the larval cuticle (puparium). This is called “pupation” andcorresponds to the pupation of other insect species. Then, the adult cuticle is formed beneath the pupalcuticle. Thus, a late pupa has three layers of cuticles. The eclosion occurs at around 95 - 105 hours
after puparium formation (APF).
In early pupae, there is only one layer of cuticle. The pupal cuticle is not yet detached from the larval
cuticle, which forms the puparium. Fat bodies become granulated and spill out when the body isdissected, making the handling a bit more cumbersome than for larvae. Also, the imaginal discs, which
are attached to the CNS, become more prominent and more difficult to remove.
1: Grasp the head of a pupa with the thicker forceps. (If you use the sharper forceps here, the pupal
cuticle of the head, rather than of the neck, will be torn apart at step 4.)
2: Stick a tip of the sharper forceps to the dorsal or lateral surface of the pupa at about one fourthfrom the head.
3: Move the sharper forceps to make a nick around the body wall. Cut apart the posterior body half
(i.e. three fourths).

- 3 -4: Put the forceps into the head cuticle (anterior one fourth) and remove internal organs from within
it. Usually the brain is located near the anterior end of the head capsule.
5: Locate the brain. Remove oesophagus and tracheae.
6: (option) If needed, exchange the thicker forceps with the microscalpel.
7: The eye imaginal discs protrude from the brain hemisphere. Leg and wing discs protrude from thethoracic ganglia. Grasp these discs, cut the nerve with either the sharper forceps or the microscalpel
to remove the CNS.
(Do not try to grasp the CNS when removing the discs. This way you would surely damage thespecimen.)* Discs may be kept until after fixation/staining.

II: Dissecting mid pupae (12-50 hours APF)
The pupal cuticle is already formed beneath the puparium. But the neck part of the body is not yet
narrowed. The leg and wing discs are already everted to form the adult legs and wings. Only thinnerves connect them with the CNS. The retina of the eye is not yet tightly connected to the body wall,
making it easy to separate the brain from the head cuticle.
1: Choose the sharper forceps.
2: Grasp the posterior part of the pupa. Open the opeculum (the lid of the puparium head) with theother pair of forceps.
3: Put the tip of the forceps into the space between the puparium (pupal case) and pupa. Remove thepuparium up to the level of the thorax.
4: Grasp the dorsal area of the anterior thorax with both forceps. Put the body apart to cut at the level- 4 -5:
6:

7:
8:of the thorax.
Open the dorsal thorax anteriorly towards the neck.
Push the anterior end of the head towards the bottom of the dish, and then squeeze the headcapsule. The internal organs of the head, including the CNS, will spill out. Remove oesophagus
and fat bodies carefully to expose the CNS. Pipetting will help to remove debris. Be careful not todamage the connecting fibres between the brain and the thoracic ganglia.
Grasp attached debris and discs, cut the region between them and the CNS. Them remove theCNS.
Cut the region between retinae and laminae.

III: Dissecting late pupae – adult (50 hours APF –)The adult cuticle is formed beneath the pupal cuticle. The head part is clearly separated from the thorax.
The neck, as well as the cervical connective that connects the brain and the thoracic ganglia, becomesvery thin. This makes it difficult to dissect the whole CNS. In many cases, it is more efficient to dissect
only the brain, or the thoracic ganglia, separately. The fully-developed compound eyes connect thebrain with the head cuticle. Removal of the brain becomes more difficult.
Adult flies secrete wax from their body, which makes them hydrophobic. Before transferring the adultflies to the dish with PBS, soak them into alcohol for 30 sec. to 1 min. to wash out this wax.

- 5 -IIIa: Dissecting the whole CNS
1: Remove the puparium case. Tear off the legs and wings with the forceps.
2: Turn the body upside down. Gently open the cuticle of the ventral thorax between the roots of thelegs. (Like the way you take off your blouse.) Cut the side wall of the thorax and remove themuscles. The thoracic ganglia will be exposed.
3: Put the forceps into the region behind the dorsal posterior head near the neck (nape). Remove thetransparent pupal cuticle like the way you take off your wig.
4: Remove the mouth part. Cut up the region between the antennae, forehead, and the top of the head.
Gently separate the right and left halves of the head capsule. (Do not put too much stress to thebrain, otherwise the optic lobes would be separated from the central brain.)
4´: Alternatively, grasp the dorsalmost region of the posterior brain with both forceps and pull thecuticle towards left and right. Open up the nick from dorsal towards ventral (via forehead andantennae.)
5: Remove oesophagus, tracheae (air sacs) and fat bodies.
6: Remove the cuticle around the neck. Take extreme care not to damage the cervical connective.

IIIb: Dissecting only the brain proper
If your purpose is to stain only the brain, it is sometimes a good idea to dissect only the brain, with thebody attached intact. The body functions as a handle so that the small brain will not be lost whenchanging the staining and washing solution. The body alias handle is removed after staining.
Legs and wings are better removed.

- 6 -C: Taking the pupa out of the puparium
1:
2:
3:
4:

Prepare black surface made of vinyl or plastic. (This is to make it easier to observe white larvae.)
Place a larva onto the black surface and paste with instant glue.
Open the lid of the head region of the puparium (opeculum). The head will be exposed.
Put the thinner (sharper) tip of the forceps into the space between the puparium and pupa. Cut theside wall of the puparium towards the posterior. (The side wall is thinner than other puparium
cuticle.) Cut the side wall a bit (one segment or two) and then open the area towards the other sideof the puparium. The pupal body will gradually be exposed.
5: The head (dorsal part and side region) and the wing discs are pushed towards the puparium cuticle.
Take extreme care not to damage the surface. For this purpose, place the thicker tip towards thepupal surface.
6: When all the body is exposed, put the forceps beneath the pupa. Since the pupa is wet, it will attach
to the forceps. Never, ever, try to grasp the pupal body!

- 7 -