To generate flies with reduced Kenyon cell numbers, we used an existing chemical method to ablate KC neuroblasts (de Belle and Heisenberg, 1994 (link); Elkahlah et al., 2020 (link); Sweeney et al., 2012 (link)). In Drosophila, 4 mushroom body neuroblasts from each hemisphere give rise to ~500 KCs each (Ito et al., 1997 (link)). Most neuroblasts pause their divisions during the first 8 hours after larval hatching (ALH), however MB neuroblasts continue to divide. Therefore, by feeding larvae a mitotic poison hydroxyurea (HU) during this time window, Kenyon cell neuroblasts can specifically be ablated (de Belle and Heisenberg, 1994 (link)). We can achieve a wide range of KC numbers by tweaking the concentration and duration of HU application (Elkahlah et al., 2020 (link)). This allowed us to generate flies with 0 to 4 KC neuroblasts, and the effect on each hemisphere was independent of the other resulting in a large range of KC numbers. However, as we described previously, the most informative brains, with intermediate numbers of KC neuroblasts remaining, are difficult to obtain—most ablation batches have a preponderance of unaffected or fully ablated mushroom bodies.
The protocol was adapted from (Elkahlah et al., 2020 (link); Sweeney et al., 2012 (link)). We set up large populations of flies in cages two days prior to ablation and placed a 35 or 60 mm grape juice agar plate (Lab Express, Ann Arbor, MI) in the cage with a dollop of goopy yeast. One day prior to the ablation, we replaced the grape juice/yeast plate with a new grape juice/yeast plate. On the morning of the ablation, we removed the plate from the cage and discarded the yeast puck and any hatched larvae on the agar. We then monitored the plate for up to four hours, until many larvae had hatched. Larvae were washed off the plate using a sucrose solution, and eggs were discarded. Larvae were then strained in coffee filters, and submerged in hydroxyurea (Sigma, H8627) in a yeast:AHL mixture, or sham mixture without HU. Ablation condition was 10 mg/mL HU, given for 1 hour. One batch experienced 15 mg/mL HU, given for 1 hour; this was done to gather more data points with a lower KC clone count. Larvae were then strained through coffee filters again, rinsed, and placed in a vial or bottle of B food (for MBON functional imaging and immunohistochemistry) or Janelia food supplemented with 0.2 mM all-trans-retinal (for behavior) until eclosion. We opened a new container of hydroxyurea each month as it degrades in contact with moisture, and we found its potency gradually declined. We achieved a U-shaped distribution of the HU effect, with many samples unaffected and many with all four KC neuroblasts lost. Ablated animals along with the control group (sham) were shipped in temperature-controlled conditions to Janelia Research Campus for behavior. A digital thermometer was kept in each shipment to record the lowest and highest temperature experienced during shipping. Batches that experienced ~15-27 Celsius were used for experiments. Animals were either shipped as larvae or late pupae, and not as adults in order to give enough time window for using the appropriate age of adult flies for behavior experiments.
The protocol was adapted from (Elkahlah et al., 2020 (link); Sweeney et al., 2012 (link)). We set up large populations of flies in cages two days prior to ablation and placed a 35 or 60 mm grape juice agar plate (Lab Express, Ann Arbor, MI) in the cage with a dollop of goopy yeast. One day prior to the ablation, we replaced the grape juice/yeast plate with a new grape juice/yeast plate. On the morning of the ablation, we removed the plate from the cage and discarded the yeast puck and any hatched larvae on the agar. We then monitored the plate for up to four hours, until many larvae had hatched. Larvae were washed off the plate using a sucrose solution, and eggs were discarded. Larvae were then strained in coffee filters, and submerged in hydroxyurea (Sigma, H8627) in a yeast:AHL mixture, or sham mixture without HU. Ablation condition was 10 mg/mL HU, given for 1 hour. One batch experienced 15 mg/mL HU, given for 1 hour; this was done to gather more data points with a lower KC clone count. Larvae were then strained through coffee filters again, rinsed, and placed in a vial or bottle of B food (for MBON functional imaging and immunohistochemistry) or Janelia food supplemented with 0.2 mM all-trans-retinal (for behavior) until eclosion. We opened a new container of hydroxyurea each month as it degrades in contact with moisture, and we found its potency gradually declined. We achieved a U-shaped distribution of the HU effect, with many samples unaffected and many with all four KC neuroblasts lost. Ablated animals along with the control group (sham) were shipped in temperature-controlled conditions to Janelia Research Campus for behavior. A digital thermometer was kept in each shipment to record the lowest and highest temperature experienced during shipping. Batches that experienced ~15-27 Celsius were used for experiments. Animals were either shipped as larvae or late pupae, and not as adults in order to give enough time window for using the appropriate age of adult flies for behavior experiments.
