Protocols to Study Behavior in Drosophila.

Drosophila melanogaster is an incredibly versatile organism capable of both innate and higher-order behaviors. These behaviors offer not only a way to assay whether or not the animal is physiologically compromised (e.g., feeding, locomotion), but also serve to assess changes in centrally mediated functions. Here we describe several high throughput, reproducible, yet inexpensive and facile behavioral assays for both larval and adult Drosophila. The larval assays all employ an agar substrate in a petri dish; the adult assays are grouped into "vial-based" and "arena-based" paradigms. While these protocols are largely designed to assess individual animals, they are sufficiently rapid that ample numbers can be tested to determine behavioral significance. Importantly, this also allows for one to control for reproductive status, age, and sex, since these factors all have a significant impact on adult behaviors. In general, it is best to designate a dedicated area for any assay, so that lighting conditions are consistent, and all animals should be tested at roughly the same time each day to minimize circadian fluctuations. Temperature and humidity should also be maintained at a constant level to minimize variability in the assays.

Functional analysis of the larval feeding circuit in Drosophila.

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Neurotrophic actions of dopamine on the development of a serotonergic feeding circuit in Drosophila melanogaster.

BACKGROUND: In the fruit fly, Drosophila melanogaster, serotonin functions both as a neurotransmitter to regulate larval feeding, and in the development of the stomatogastric feeding circuit. There is an inverse relationship between neuronal serotonin levels during late embryogenesis and the complexity of the serotonergic fibers projecting from the larval brain to the foregut, which correlate with perturbations in feeding, the functional output of the circuit. Dopamine does not modulate larval feeding, and dopaminergic fibers do not innervate the larval foregut. Since dopamine can function in central nervous system development, separate from its role as a neurotransmitter, the role of neuronal dopamine was assessed on the development, and mature function, of the 5-HT larval feeding circuit. RESULTS: Both decreased and increased neuronal dopamine levels in late embryogenesis during development of this circuit result in depressed levels of larval feeding. Perturbations in neuronal dopamine during this developmental period also result in greater branch complexity of the serotonergic fibers innervating the gut, as well as increased size and number of the serotonin-containing vesicles along the neurite length. This neurotrophic action for dopamine is modulated by the D2 dopamine receptor expressed during late embryogenesis in central 5-HT neurons. Animals carrying transgenic RNAi constructs to knock down both dopamine and serotonin synthesis in the central nervous system display normal feeding and fiber architecture. However, disparate levels of neuronal dopamine and serotonin during development of the circuit result in abnormal gut fiber architecture and feeding behavior. CONCLUSIONS: These results suggest that dopamine can exert a direct trophic influence on the development of a specific neural circuit, and that dopamine and serotonin may interact with each other to generate the neural architecture necessary for normal function of the circuit.