Flys do not naturally express ATP receptors, they are not encoded in the D. melanogaster genome. Neurons that have been made to express the ionotropic purinoceptor P2X2 (Valera et al. 1994), i.e. under the control of GAL4, can be stimulated by ATP. ATP can be delivered locally using a micropipette loaded with ATP in solution, which it inserted into the brain to target a loci of interest.

The Miesenböck group first did this using caged ATP that was photo-released by widefield UV illumination (Lima et al.) . The Wilson group used P2X2 and pressurized local ATP delivery to get specific responses from neurons (Liu et al. 2012). ATP has also been delivered using electrophoresis (Handler et al. 2019), which we have yet to try.

Executing pressurised ATP delivery#

You will typically want a fly on your rig expressing one fluorophore under the control of a genetic driver, e.g. GFP. The brain should be exposed and IR light directed at the brain (see dissection guide here). We will aim to see the delivered ATP using a different fluoropore, i.e. an RFP.

Equipment:#

  1. Microscope, e.g. Olympus BX51WI
  2. You will need a longpass filter cube for visualising GFP+IR (i.e. your neurons), and a second lonpass filter cube for visualising RFP+IR (i.e. the ATP solution).
  3. PV 800 Pneumatic PicoPump
  4. This will connect to your DAQ
  5. Micropipette holder
  6. MP-225
  7. With XYZ controller
  8. Mechanical turret for micropipette holder
  9. IR light source
  10. Freshly pulled glass pipette
  11. Made with program 56.
  12. Glass pipettes pulled to 7-10 MΩ

Reagents:#

  1. ATP disodium hydrate salt, CAS: 34369-07-8, MW: 551.14, SigmaAldrich: A2383-25G
  2. Alex Fluor 555 hydrazine, MW: 1150, Invitrogen A20501MP, 1 mg
  3. Lab saline

ATP solution#

  1. 10 mM ATP disodium in saline, approx. 1 mM Alex Fluor 555 hydrazine
  2. Master solution: 1 ml, 1 Eppendorf tube, keep at -80C, i.e. 1. 5.5 mg ATP 1. 1 mg fluorophore 1. 1 ml saline. Cool saline to just above 0C, otherwise ATP may convert to ADP! 1. Storage: -20C or lower, protect from light
  3. Aliquot: i.e. 1 experiment each, min of 5 ul in each tube 1. Into PCR tubes, 5 ul in each, or 600 ul Eppendorf tubes. No further dilution needed. 1. Storage: -20C or lower, protect from light
  4. If you need to alter, consult the molarity calculator. If you want to do bath delivery you can dispense with the fluorophore and create a 10 mM solution as 1.1g ATP in 200 ml of saline.
  5. Process:
  6. Take a 1.5 ml Eppendorf tube, put it on ice
  7. Fill with 1 ml of saline
  8. Take an empty box for your aliquots, put on dry ice with either PCR tubes or 600 ul Eppendorf tubes
  9. Measure out your ATP powder. Add to saline solution. Mix well.
  10. Open the Alexa Fluor tube, add the 870 ul of the 1 ml solution . Mix well.
  11. Get a 3 ml syringe and a filter bit, Millex-GV SLGV004SL. Open syringe carefully, remove plunger, fasten to filter bit. Filter into a new 1.5 ml Eppendorf tube.
  12. Add solution to syringe tube with pipette. Replace plunger and slowly apply pressure until solution filters through. There will be some volume loss.
  13. Put on ice
  14. Now with a p20 (or p200 if doing larger) aliquot into your smaller tubes, that are sitting on dry ice. E.g. add 20 ul to each 600 ul Eppendorf tube.
  15. Storage: -20C or lower, protect from light

Stimulation paradigm#

  1. Stimulations to try:
  2. Single pulse of 10 ms at a pressure of 6 psi.
  3. Train of 5 ms pulses, interspersed at 20 ms, at a pressure of 6 psi.

Process#

Troubleshooting#

  1. GTP does not activate P2X2 and could be used as a control.

Cons#

  1. Leak from the P2X2 receptors when they are overexpressed could impact animal behaviour and longevity. When expressed panneuronally, flies appear less coordinated in their movement.
  2. Pressurised delivery may mean you have perfusion limited latencies.
  3. If ATP is released at 6 psi for more than10 ms it may effect control neurons not expressing P2X2.
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