Distribution of functional glutamate receptors in cultured embryonic Drosophila myotubes revealed using focal release of l-glutamate from caged compound by laser

During the formation of neuromuscular junctions in Drosophila embryos, glutamate receptors undergo a drastic change in distribution. To study the underlying mechanism of this developmental process, it is desirable to map the distribution of functional receptors with accurate spatial resolution. Since glutamate receptors desensitize within several milliseconds, the agonist must be applied rapidly. To fulfil these requirements we used laser stimulation of a caged compound to release

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-glutamate at a focal spot. Since the glutamate receptor channel is permeable to Ca²⁺, we assayed the change in internal Ca²⁺ concentration using a Ca²⁺ indicator, fluo-3. Using this approach, we mapped the distribution of functional glutamate receptors in cultured embryonic Drosophila myotubes and myoblasts. Consistent with previous immunofluorescence studies using an antibody against a glutamate receptor subunit, a large increase of internal Ca²⁺ concentration was observed when laser stimulation was located close to some nuclei in the myotube. No change was detected when the laser stimulus was applied over any regions of the myoblasts. No increase of the internal Ca²⁺ concentration in myotubes was observed when the external solution contained either glutamate at a desensitizing concentration (1 mM) or a glutamate receptor channel blocker, argiotoxin (1 μg/ml). These results indicate that a rise in intracellular Ca²⁺ concentration can be used to show the distribution of the functional receptor on the muscle surface membrane.