For both mammals and invertebrates, developmental EX 527 concentration perturbations of neurotransmitter metabolism can have neuroanatomical sequelae (Budnik et al., 1989, Lawal et al., 2010 and Levitt et al., 1997). We therefore analyzed the morphology of the MBs and CCX in the prt1 mutant and found it grossly intact in paraffin sections of adult brains stained with hematoxylin and eosin staining (H&E; Figure 4D; data not shown). To rule out more subtle neuroanatomical changes, we performed volumetric analyses of the MB calyx and CCX (ellipsoid body + fan-shaped body). We detected no difference in either calyx or CCX volume between CS and prt1 ( Figures 4E and 4F),

indicating that prt1 does not result in significant anatomical defects. To further examine changes in the function of the MBs and other tissues expressing PRT, we investigated PI3K inhibitor prt1 mutant behavior. We first outcrossed the prt1 flies for six generations into the wild-type strain CS. Outcrossing removed a closely linked mutation that reduced viability and fertility (data not shown), and all behavioral experiments were performed using the outcrossed lines. The outcrossed prt1 flies were viable, fertile, and showed no obvious external morphological defects. The relatively high level of PRT expression in the MBs, as compared to other structures, suggests that it may play a

role in olfactory classical conditioning, which is known to require the MBs (Davis, 2011). We used a modified T maze to test olfactory classical conditioning, as previously described (de Belle and Heisenberg, 1994). As controls for these experiments, we first established that prt1 flies had normal avoidance of both electric shock and the odors used to test learning ( Figures 5A–5C). We next tested olfactory learning. We found that prt1 mutants have a learning defect, evidenced by a decreased performance index immediately following training ( Figure 5D). The performance indices for prt1 were also reduced at 30 min and 6 hr after training ( Figure 5D). The difference between CS and prt1 was consistent over short-term (30 min)

and middle-term (6 hr) phases of memory, suggesting normal memory decay in prt1 of flies. We next tested prt1 behavior using several other well-established assays. Performance indices for negative geotaxis and fast phototaxis were equivalent to those of wild-type flies ( Figures S3A and S3B), indicating that gross locomotor activity and the response to both mechanical stimuli and visible light are intact in prt1. We did find a modest impairment in courtship behavior. Although prt1 males performed all of the necessary courting rituals, they spent less time courting ( Figure 6A). In the course of performing courtship assays, copulation was observed. Normally, males mount the female from behind, curling their abdomen upwards to allow coupling.