Every now and then, a seminal paper really leaves its mark. One of the papers that most impressed me during the 1990s was an early use of degenerate PCR to identify a massive set of olfactory receptors in rat, and thence to map their distribution within the nose. In this single paper (much imitated since), Axel and Buck cut through generations of speculation on the molecular basis of olfactory perception, providing a simple answer. And now Axel has resurfaced in a Cell paper published this January that actually images synaptic activity in live fly brain, through its proxy, calcium, when the fly is exposed to different odours. This is hardly an ambulatory,non-invasive procedure: the fly was decapitated, its head mounted in agar, and the cuticle dissected back to expose the brain. Nor is it technically simple:multi-photon confocal microscopy is still more in the realm of physics research than a routine biological tool. Nonetheless, it proved possible to expose antennal lobes to different compounds, and to image the response in deep structures of the brain, for up to five hours. In the longer term,development of these procedures might produce new, less invasive and more physiological access to brain function.
Real-time calcium measurement in genetically defined cells in Drosophila is not new. Previous studies have used the transgenic jellyfish calcium reporter, aequorin, and, more recently, calcium-sensitive green fluorescent protein (GFP) derivatives, such as G-CaMP and pericam. In common with aequorin, these calcium reporters are proteins. This means that their expression can be directed to specific cells within a particular tissue using the beautiful genetic tools unique to Drosophila.
In this paper, the gene encoding G-CaMP was placed under the control of a transcriptional promoter that is activated by the GAL-4 transcription factor,and transgenic flies made. These flies have the potential to make G-CaMP protein in any cell in which GAL4 is expressed. Then they constructed a second set of flies, where GAL4 transcription was controlled from olfactory-lobe-specific promoters. When the two strains were crossed, high levels of G-CaMP were thus confined to the lobes. However, the lobes are buried quite deep in a relatively large (by Drosophila standards)structure, so conventional epifluorescence or even confocal microscopy would not suffice to give a good signal. Instead, the authors used two-photon confocal scanning, which allows longer illuminating wavelengths, and thus deeper tissue penetration to visualise the G-CaMP-mediated calcium signal.
The output of these techniques was remarkable: the authors were able to show that particular odorants elicited responses in particular lobes (or combinations of lobes) and that these mappings persisted between individual flies. By directly stimulating the antennal nerve, the authors were also able to show that the calcium responses they were measuring were a direct function of spike frequency in the antennal nerve.
Thus, this study nicely complements the original Axel and Buck paper, which mapped out the other end of the sensory pathway and the distributions of the odorant receptors in the nose. Axel's current work also shows that calcium in the olfactory lobes could be taken as a faithful correlate of electrical activity in neural tissue.