Immunocytochemical study of a temperature‐sensitive choline acetyltransferase mutant of Drosophila melanogaster

Using a monoclonal antibody to choline acetyltransferase (ChAT), we have identified immunoreactive synaptic terminals in the neuropil regions of the cephalic ganglion of Drosophila melanogaster . This study demonstrates the distribution of antibody‐labeled structures within the optic lobe, and then investigates the immunoreactivity altered by mutation in two temperature‐sensitive ChAT alleles, cha ts−1 and cha ts−2 . The general structure of the optic lobe was first observed by means of the silver impregnation technique. Then the presence of ChAT immunoreactivity was determined by the application of antibody [1G4] conjugated with HRP to frozen sections, followed by the 3,3′‐diamino‐benzidine tetratinct layers, which correspond to the three synaptic layers of the laminarneurons, in the medulla. Also, staining appeared in four distinct layers in the lobula. In addition, weaker staining was observed in the lamina, which corresponds to the retinula cell terminals. Somal layers were not stained. In Canton‐S (wild‐type), the three medullar layers stain distinctly at both 19°C and 30°C. In cha ts−1 at 19°C, the stain appeared in the same layers as that of Canton‐S, but with somewhat lower density. In cha ts−2 at 19°C, the density of the stain was even lower. The densities of the stain in these mutants were further decreased after exposing the flies to 30°C. The decreases were dependent on the length of exposure to the higher temperature. The decrease in stain of the specimens obtained after 24 hours exposure to 30°C was clearly recognizable in both cha ts−1 and cha ts−2 . The stain was visually unrecognizable after 120 hrs incubation at 30°C in cha ts−1 , and after 80 hrs in cha ts−2 . The stained structures in the medulla are apparently terminals of the laminar neurons. α‐Bungarotoxin (α‐BTX) conjugated with HRP and visualized by the DAB‐H 2 O 2 reaction was also used to stain frozen sections of wild‐type and mutant Drosophila. The three layers of α‐BTX‐HRP stained structures in the medulla overlapped with the layers revealed by 1G4. Exposure to high temperature (30°C) for up to 120 hours did not show any effect on the stain obtained by α‐BTX‐HRP in either allele. These results show that 1G4, a monoclonal antibody specific for Drosophila ChAT, can be used to identify ChAT‐containing neurons. In the temperature‐ sensitive ChAT mutant, cha ts , the reduced immunoreactivity paralleled the reduced ChAt activity levels which have been observed in this mutant.