Dap160^EMS1/Dap160^Δ2 third instar larvae have supernumerary boutons at the neuromuscular junctions of muscles 6/7 and 4 compared to controls.

The levels of membrane uptake (dye internalisation) in Dap160^Δ1/Dap160^Δ2 mutant synaptic boutons in response to nerve stimulation is significantly reduced compared to controls. Increased uptake is seen when these flies are treated with chlorpromazine.

Homozygotes die as third instar larvae. Neuromuscular junction (NMJ) morphology is abnormal in Dap160^Δ1/Dap160^Δ2 third instar larvae; boutons are irregularly spaced and large boutons are surrounded by smaller satellite boutons. Bouton number normalised to muscle area is significantly increased in Dap160^Δ1/Dap160^Δ2 larvae (3.5 +/- 0.07 μm^-2) compared to controls (1.7 +/- 0.07 μm^-2). In addition, the boutons show more frequent branching than controls; the larval NMJ has 26.2 +/- 2.1 branch points per synapse in Dap160^Δ1/Dap160^Δ2 animals compared to 5.1 +/- 0.3 branch points per synapse for controls. In 5mM Ca²⁺ at 22^oC, the amplitude of the excitatory junctional potential (EJP) at the neuromuscular junction (NMJ) in Dap160^Δ1/Dap160^Δ2 third instar larvae is not different from that of controls. The EJP amplitude at the NMJ of Dap160^Δ1/Dap160^Δ2 third instar larvae is normal in 5mM Ca²⁺ at 34^oC. In 0.5 mM Ca²⁺, the EJP amplitude at the NMJ of Dap160^Δ1/Dap160^Δ2 third instar larvae is normal at 22^oC, and is slightly reduced compared to controls at 34^oC. In 0.5mM Ca²⁺, the frequency of miniature EJPs (mEJPs) at the NMJ is increased more than 2 fold in Dap160^Δ1/Dap160^Δ2 larvae compared to controls at 22^oC. mEJPs with an unusually large amplitude are seen at both 22^oC and 34^oC. Dap160^Δ1/Dap160^Δ2 larvae show a mild defect in endocytosis at 22^oC (assayed by dye uptake in third instar fillet preparations), which is exacerbated at 36^oC. Dap160^Δ1/Dap160^Δ2 larvae show a stimulation-dependent decrease in EJP amplitude when stimulated at 10Hz. This decrease is more severe at 34^oC than at 22^oC. Ultrastructural analysis of the NMJ shows that the number of vesicles is reduced to about 60% of controls in Dap160^Δ1/Dap160^Δ2 larvae at 22^oC without stimulation. The number of vesicles is further reduced to about 23% of controls upon stimulation in 60mM K⁺ at 34^oC. The number of active zones is not reduced in the mutant larvae compared to controls, but there is a reduction in the number of vesicles at the active zones in mutant larvae at 22^oC without stimulation and a further decrease upon stimulation with 60mM K⁺ at 34^oC. There are an increased number of vesicles with large diameter in the mutant synapses. Endocytic intermediates, mostly consisting of collared pits and Ω structures are seen in the mutants (these structures are rarely seen in controls). The collared pits are seen at both the active zones and in periactive zones in the mutant larvae.