Flies expressing Chc^{N.4C.T:Zzzz\FLAG,T:Zzzz\TC} in a Chc¹ mutant background are viable. They display no behavioural defects, the morphology of the third instar larval neuromuscular junctions is indistinguishable from controls, postsynaptic receptors are clustered normally and neurotransmitter release in response to low and high frequency nerve stimulation is similar to controls. These phenotypes are seen both in the presence and absence of Chc photoinactivation by FlAsH-FALI.

When flies expressing Chc^{N.4C.T:Zzzz\FLAG,T:Zzzz\TC} in a Chc¹ mutant background are pretreated with FlAsH-FALI to photoinactivate Chc, they show similar amounts of membrane uptake (dye internalisation) to controls in response to nerve stimulation. However, unlike in controls, the dye is often concentrated in subsynaptic structures, rather than the typical donut shape. A similar phenotype is seen when flies are treated with chlorpromazine, but the phenotype is not enhanced by combining treatment with FlAsH-FALI and chlorpromazine together. When flies are stimulated for a second time in the absence of dye, the previously internalised dye is not unloaded, indicating that membrane internalised in the absence of Chc is not released.

The synaptic boutons of third instar larvae expressing Chc^{N.4C.T:Zzzz\FLAG,T:Zzzz\TC} in a Chc¹ mutant background and subjected to FlAsH-FALI to photoinactivate Chc are almost devoid of synaptic vesicles and show giant membrane invaginations, with some measuring >1μM in cross section. There is a decrease in vesicle number per area and an increase in vesicle size. The remaining round or oval shaped vesicles in these boutons show heterogeneity in size and a population of larger vesicles and cisternae is observed. However, active zones mitochondria and subsynaptic reticulum are all present in both control and FlAsH-treated synapses.

FlAsH-FALI treatment has no effect on the excitatory junction potential (EJP) amplitude seen in flies expressing Chc^{N.4C.T:Zzzz\FLAG,T:Zzzz\TC} in a Chc¹ mutant background during low frequency stimulation. The same result is seen at all Ca[2+] concentrations tested. However, when EJPs are recorded during intense stimulation, FlAsH-FALI photoinactivation reduces the relative EJP amplitude compared to controls, with the amplitude dropping to <25% of the initial value.

When flies expressing Chc^{N.4C.T:Zzzz\FLAG,T:Zzzz\TC} in a wild type background are pretreated with FlAsH to photoinactivate Chc, levels of membrane uptake (dye internalisation) are similar to controls, but unlike in controls, the dye is often concentrated in subsynaptic structures, rather than the typical donut shape.

Flies expressing Chc^{4C.T:Zzzz\FLAG,T:Zzzz\TC} in a Chc¹ mutant background are viable and don't show any obvious developmental defects. No defects are seen in climbing, negative geotaxis or flying. Under normal conditions electroretinogram recordings in response to a light flash are comparable to wild type. However when FlAsH is micro-injected under the photoreceptor layer these flies lack the normal "on" and "off" transients seen in controls.

Flies expressing Chc^{4C.T:Zzzz\FLAG,T:Zzzz\TC} in a wild type background do not show any defects in climbing, negative geotaxis or flying. Electroretinogram recordings seen in response to a light flash are comparable to wild type.