BRAIN DISSECTION FROM ADULT DROSOPHILA **The initial steps for this dissection are the same for the wholemount dissection and eye/cuticle decolorising protocol available from the same source as this file. A diagram is also available and recommended (dissect.gif)** Required: Dissecting dish (5-10cm tissue culture dishes) containing approx 5mm depth of polymerised Sylgard (BDH) or similar transparent silicon rubber. Fix - 4% paraformaldehyde in PBS (10ml), prepared fresh or from a frozen 20% stock (paraformaldehyde does not dissolve readily and can be dissolved by heating (60 C over a long period - do not boil), or by alkali addition and re-pH'ing (see Ashburner's Drosophila book) Dissecting pins (as fine as possible - 'minuten pins') Very sharp forceps (A strongly recommended sharpening stone is the "original Arkansas"). New forceps are NOT sharp. ================================================================= SUMMARY - based on the diagram dissect.gif PLEASE read the full description for tips !!! Pin the fly ventral side up through the abdomen and remove all six legs (diagram B). As shown in diagram A: * = perform bilaterally 1. Break the cuticle at 'a' * 2. Tear the cuticle along line 'b' (superficially) 3. Tear the cuticle along line 'c' * (easiest by 'pinching') 4. Hold the projection 'd' and lift carefully until the cuticle loosens, then lift the entire cuticle piece by pulling at the anterior edge of the loosened piece (by 'a'), the VNC should now be exposed (very little practice needed). 5. remove the trachae exposed at the posterior-most point (under 'd') and break the abdominal nerve which is then exposed using two pairs of forceps. 6. Holding the abdominal nerve, lift the ganglion, clearing nerves underneath with a gentle, slow sweep of a second pair of forceps. The VNC should now be attached only at the neck. 7. slide one point of each of two forceps in under the cuticle at 'e' and, holding the more posterior pair steady, tear the cuticle with a lateral movement 'shelling' the eye cuticle away. * Remove the head capsule as one piece. 8. grasp the 'neck' cuticle and tear the VNC, brain (and normally gut) away, still attached to the cuticle. 9. using motions tangent to the CNS, and without touching the CNS (except occasionally with the side of the forceps), clean the prep of the remaining cuticle and trachae. 10. Stop the timer =:^> Diagram C shows the approximate position of the various parts of the CNS as they relate to the whole body of the fly. =================================================================== Full Method: Flies are anaesthetised and dipped in 95% ethanol in order to reduce their surface tension in aqueous medium, then transferred into fix in the dissecting dish. The fly is pinned through the abdomen (ventral side uppermost) to the Sylgard surface, head towards you. The front four legs are removed close to their insertion into the thorax. Gently tilting the head towards you and extending the proboscis (if necessary), you will see a transparent skin of cuticle between the proboscis and neck. break this cuticle by pinching it SHALLOWLY and pulling. The head will (usually) now stay tilted towards you. You will be able to see silvery tracheae in the bottom half of the head (closest to you), one on each side of the midline, behind the base of the proboscis, extending for about half of the visible head capsule. Insert the forceps and grab the tracheae. The brain is contained in the other half of the head capsule (away from you), so you can dig quite deeply without damaging it. Also, none of the major cerebral nerves are likely to be damaged from this insertion vantage. Remove all or most of the tracheae (a pinch followed by a gently tug often removes the whole air bubble in one go). LEAVE the tubular eye tracheae (which may be visible) as attempting to remove these can easily damage the retina. Your fly should now look like diagram A. a) On the thorax, two thin, darker lines of cuticle will be apparent (immediately posterior and lateral to the front leg holes). Using one pair of forceps to hold one of the lines (deeply, but as lateral as possible) grab the cuticle lateral to this point with the second pair and tear sideways (away from the midline) until the line is broken. Don't remove any cuticle. Repeat for the other side. b) Superficially pinch and tear away the cuticle between the two front leg holes. c) Similarly, break the lighter cuticle at the same lateral position on both sides of the thorax as far back as the second leg hole (Do not work more medially than the outermost edge of the leg holes.) d) Grasp the cuticle between the two T2 leg holes (again superficially) and gently/slowly pull up and towards you until you can see that it has separated (DO NOT pull too far up as the ganglia will distort). Grasp the cuticle between the T1 leg holes (.......superficially) and pull up and away from you. The ventral cuticle should be removed easily to expose the T1 and T2 lobes of the ventral ganglia. At the most posterior margin of this hole you should see a tubular trachea underneath where the T2 leg holes were. This trachae covers the T3 and part of the abdominal neuromere and can be removed with extreme caution, remembering that the nervous tissue is directly below it. After removal of this trachae, and (for convenience) part of the cuticle posterior to it, the thick main abdominal nerve can be seen, no attempt should be made to move the VNC before breaking this nerve as distortion or complete dissociation of the abdominal neuromeres will result. Break the abdominal nerve by holding it firmly with two pairs of forceps and moving the more posterior pair to tear the nerve. Gently slide one pair of forceps under the VNC and break the smaller nerves anchoring it to the dorsal musculature by gradual lifting until the VNC is only anchored at the dorsal end (at the 'neck' of the fly). e) Slide one 'leg' of a pair of forceps superficially under the cuticle at the posterior margin of the eye. Place another leg of another pair of forceps more posterior to these and grasp the cuticle. Moving the forceps closest to the eye in a direction distal to the fly and away from you should 'shell' the eye cuticle away from the optic lobe. Leaving the cuticle attached, repeat for both sides. Break the cuticle behind the brain and remove the head capsule. If you now grasp the cuticle near the 'neck' (avoiding the neck itself), and tear away from the fly, the cuticle should bring the CNS with it. This cuticle can now be removed by grasping it with forceps at two points and tearing the cuticle between these points with all of the force applied at a tangent to the brain. NEVER touch the brain with the tips of your forceps, you WILL damage it, always hold the cuticle/trachae attached to the brain and manipulate these. If you see the brain distorting AT ALL, stop and try pulling at two different pieces of cuticle. Normally, the gut will also be attached, this should be pulled through the sub-oesophageal foramen from the thoracic direction whilst steadying the brain on both sides with the shafts (not tips) of an open pair of forceps. The last (small) pieces of cuticle and trachae can also be removed in this way although the risk increases the more you touch the brain, so indirect tearing is always recommended. That's it, you've got a clean brain, hopefully in a reasonably short time (very little practice is needed if you follow the tips given, but a steady hand is a major bonus and I wouldn't even attempt it after a few pints unless you want Drosophila puree.) Sean May - 1993 lsrei@csv.warwick.ac.uk ys@dna.bio.warwick.ac.uk