Metamorphosis is a stressful time in a frog's life; not only does the tadpole develop legs and lose its tail, but it also has to cope with the replacement of its gills with lungs. This transition from water-breathing to air-breathing coincides with a `switch' in the animal's response to plummeting oxygen levels: while tadpoles respond vigorously and start breathing faster,frogs are relatively insensitive and tend to breathe more slowly when oxygen levels drop. How is this developmental switch regulated? To find out,neurobiologist Richard Kinkead and his students at Université Laval in Québec, Canada, took a closer look at the neurons in a frog's developing brain (p. 3015).

The team suspected that the neurotransmitter noradrenaline, which is known to influence breathing rhythms in mammals, might play a role in the different responses of frogs and tadpoles to falling oxygen levels. To see if noradrenaline mimics the developing frog brain's response to low oxygen levels, Kinkead's student Stéphanie Fournier carefully removed the brainstems of tadpoles and adult bullfrogs and bathed the brainstems in noradrenaline. Using suction electrodes, the team recorded the bursts of brainstem activity that would drive breathing movements in a live animal. As the team had suspected, brainstems bathed in noradrenaline responded just like brains exposed to oxygen-depleted bathing solution; tadpoles' brainstems showed increased bursts of activity, while those of adult frogs decreased their activity.

Noradrenaline binds to different kinds of receptors, calledα-adrenergic receptors, in the brain. `We wanted to examine which specific receptors are involved in the switch,' Kinkead says. When the team added blockers of α1 and α2 adrenergic receptors to the solution bathing the brainstems, they saw that blocking either of these receptor types prevented the normal response to low oxygen levels in tadpole brains, but only α1 blockage prevented the normal response of frog brains to oxygen depletion. This suggests that although activation of any α-adrenergic receptor mediates tadpoles'responses to falling oxygen levels, only α1 receptor activation mediates the breathing responses of adult frogs in oxygen-depleted conditions.

`But these findings still didn't explain why the response to low oxygen levels changes during development,' Kinkead says. His attention was drawn to the neurotransmitter γ-aminobutyric acid (GABA). He knew that, in mammals, activation of GABA-releasing (GABAergic) neurons has excitatory effects early in development, but exerts inhibitory effects later on. So Kinkead reasoned that `GABAergic neurons are a good candidate to explain the developmental switch seen in frogs.' To determine whether GABAergic neurons act as the `middle man' in frogs' developmental changes, the team added chemical blockers of GABA to the brainstem bathing solutions to see whether this would prevent the typical responses of frogs and tadpoles to low oxygen levels. They saw that blocking this neurotransmitter before adding noradrenaline to the bathing solution prevented the traditional response to low oxygen in both tadpole and frog brains. The team concluded that activation of GABA-producing pathways is necessary for noradrenaline to exert its effects on the brain when oxygen levels fall. `These results allowed us to pinpoint a mechanism for the developmental switch,' Kinkead says, reasoning that maturation of these pathways during development accounts for the switch.

Taken together, the findings shed light on the transition from water- to air-breathing. `Bits and pieces of information were already there on the table,' Kinkead says. `What's interesting about this study is that it puts various parts of the puzzle together.'

Fournier, S., Allard, M., Roussin, S. and Kinkead, R.(
). Developmental changes in central O2chemoreflex in Rana catesbeiana: the role of noradrenergic modulation.
J. Exp. Biol.