We're not the only creatures that feel the detrimental effects of having too much to drink – ethanol sends flies and rodents to sleep as readily as humans. Tieqiao Wen and colleagues at the University of Georgia wondered what modulates the effects of ethanol on the Drosophila central nervous system and whether any specific neurons are involved. They concentrated their efforts on a signalling molecule called neuropeptide F(NPF) and its receptor NPFR1. The equivalent molecule in mice is known to affect sensitivity to ethanol, so would neuropeptide F have the same effect in flies?
First, the team stained the brains of adult fruit flies to see where they could find neurons expressing neuropeptide F. They found that the two pairs of neurons strongly expressing neuropeptide F had extensive branches, covering areas of the brain important for regulating feeding, flight and locomotion;all potential targets for regulation by neuropeptide F.
Next, the team created mutants from two different lab strains of fly,knocking out neuropeptide F neurons in fruit fly larvae. When these larvae reached adulthood, the team compared the behavioural response of normal and mutant flies to ethanol exposure. To do this, they placed flies in a jar with a swab soaked in ethanol, and counted the number of flies that had fallen asleep at the bottom of the jar every 5 minutes. They used a concentration of 42% ethanol in most experiments as it reliably sends flies to sleep, but the toxic effect is minimal. In 42% ethanol, the team noticed that all the non-mutant flies were asleep after 65 minutes of exposure, whereas 60% of the first mutant strain and 42% of the second mutant strain were still wide-awake at the top of the jar. This shows that neuropeptide F is likely to play a key role in how sensitive flies are to a dose of ethanol; normal flies with intact neuropeptide F neurons clearly felt alcohol's sleep-inducing effects.
The team observed similar effects when they knocked out NPFR1 signalling– mutant flies that lacked the neuropeptide F receptor were more resistant to the effects of ethanol and, just like the two strains of neuropeptide F mutant flies from the first experiment, recovered more quickly from the effects of ethanol than normal flies.
However, the team needed to be sure that what they were seeing was not due to a secondary effect of knocking out neuropeptide F neurons at an early stage of development. To test this, they used a temperature-sensitive mutant fruit fly, in which the team could block transmission of neuropeptide F-containing neurons at a certain temperature. Sure enough, these mutant flies were more immune to the effects of ethanol only when the team blocked neuropeptide F neuron transmission, showing that neuropeptide F is indeed the key to how ethanol affects fruit flies. The team wondered, in that case, what would happen if neuropeptide F was over-expressed? The result was as they expected:flies over-expressing neuropeptide F were even more likely to fall asleep when the team exposed the flies to ethanol.
The fruit fly NPF-NPFR1 pathway is important as an invertebrate model of how ethanol affects neurons, because of its similarity to a mammalian pathway and also because it is ethanol-specific. Indeed, at low concentrations,ethanol attracts Drosophila to food, something to remember next time you have an over-ripe piece of fruit in your kitchen.
