Functional imaging of spontaneous activity in the Drosophila brain Mushroom-Bodies and their circadian regulation – FlyBrainImaging

The main approach of this project concerns the in-vivo recording of brain activity, in continuous, over the long periods, as over-night and even up to 48 hours, using the bioluminescence technique (GFP-aequorin) that we have recently developed in my laboratory. We are studying principally the Mushroom-Bodies, a brain structure implicated in the olfactory learning and memory and the sleep, as well as the neurons implicated in the circadian rhythms (ventro-lateral neurons, dorsal neurons, etc.). This approach, mainly neurophysiologic, is complemented by multiples genetic approaches (tools) provided by Drosophila, as the several mutations and the directed expression of specific RNAi or else, various effector genes allowing to activate or inhibit a specific group of neurons. Technically, with the novel bioluminescent recording setup, we can record the calcium activity at a temporal resolution of 10 msec, and so over several hours. In addition, in parallel, we have developed a device allowing to quantify the locomotor activity of the fly (through the quantification of the rotation of a small ball maintained by a little airstream) simultaneously of the brain activity. In such a way, we would like to correlate the calcium activity of the various brain structures with a behavior, as the locomotor activity. Finally, more recently, we record, by video-tracking, the locomotor activity of the flies, permitting to evaluate the sleep phases of the flies.

First, I would like to remind that the in-vivo functional brain imaging is a delicate, tricky and heavy technique, and consequently it requires a certain period of training to be successful and efficient. In my laboratory (J.R. Martin), as planned in the initial project, we have characterized the different parameters of the spontaneous peak of Ca2+-activity in the Mushroom-Bodies (MBs), during the night. Briefly, the flies presents two peaks of activity in the MBs, a short peak of about 3 minutes duration with a high amplitude, and a second peak, much longer (about 3 hours), but of weak amplitude (see publication: Minocci et al., BBA-MCR, 2013). Moreover, as the MBs are well known to be involved in the regulation of the sleep, for the moment, our first preliminary results tend to show that the these peaks are disturbed in flies mutated for some genes known to affect sleep, as minisleep. In parallel, behavioral analysis performed in video-tracking, which allows to precisely quantifying the sleep, reveal that some mutants (or targeted specific RNAi in the MBs) present some sleep distortion, as for example, defaults in “falling in sleep”.
For the F. Rouyer experiments, A. Chatterjee (Post-Doc) has benefited of the knowledge and the expertise of my laboratory, acquired since few years, to record the calcium activity of divers groups of neurons implicated in the circadian rhythms. They have started by the recording of the clock neurons (spontaneous activity in constant darkness) with different P[Gal4] drivers. The preliminary results suggest a calcium activity at the morning in the neurons responsible for the morning activity. They have also showed, using a temperature sensitive system to activate the neuronal activity, that the activation of some clock neurons generates activity in the MBs. These original results represent a first step toward the characterization of the output pathways of the circadian neurons.

In perspectives, as planned in the initial project, we will pursue the planned experiments. We will continue the physiological characterization and the search of the functional role of these peaks of activity observed in the MBs during the night (sleep and/or memory). Moreover, recently we have noticed that the degree of oxygenation of the flies seems to play a role on these peaks. Dedicated experiments to study specifically this effect are now in preparation. In parallel, we are recording the behavioral activity of the flies by video-tracking in a light/dark cycle to quantitatively and qualitatively analyze the sleep.
F. Rouyer laboratory will pursuing the characterization of the activity of different circadian neurons and of the putative networks, as well as the identification of the output (efferent) clock neurons.

Daiana Minocci (Post-Doc) and Pierre Pavot (A.I.) have presented the first results at the annual meeting of the “French Invertebrates Neurobiology Club”, at Paris, May 24-25, 2012, and more recently, P. Pavot at the same meeting, in Lyon (June 13-14th, 2013). Moreover, an article describing the first characterization of the nocturnal peaks of activity in the Mushroom-Bodies has been published in BBA-MCR, July, 2013. [Minocci, D, Carbognin, E Murmu, M, Martin, JR (2013). In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach. Biochim Biophys Acta., Mol. Cell Res., 1833, 1632-1640.

Recording neuronal activity in-vivo is required to understand how the brain controls behaviour, but it faces important technical difficulties, in particular in invertebrates. Several optical imaging techniques, based on calcium activity using fluorescent markers, have been developed to study neuronal activity and the neural code underlying major neurophysiologic functions. However, fluorescence requires light excitation that induces autofluorescence, phototoxicity and photobleaching, which limit the use of fluorescence to superficial structures, and for recordings in the range of seconds or minutes. Consequently, the recordings of the activity of most brain structures, over long periods (such as hours or days), remain difficult.
A new bioluminescence calcium imaging approach, based on a chimeric GFP-aequorin gene, has recently been developed, allowing to record in-vivo neuronal activity in brain circuits. We have generated transgenic Drosophila, and targeted GA to different brain structures with the P[GAL4] expression system to record spontaneous or induced activity. Signals have been recorded from the Mushroom-Bodies (MBs), mainly known for their role in olfactory memory and sleep, as well as from the Central Complex, that is involved in the regulation of locomotor activity. Expressing GA in all brain neurons allowed to record spontaneous or induced activity of the whole brain, over several hours. These results (Martin et al., PLoS-ONE, 2007) demonstrate the capability of this new technique to record any part of the brain in-vivo. Preliminary data obtained from long term recordings of spontaneous activity (in the best case, up to 48 hours), reveal that the MBs exhibit a single and massive peak of Ca2+-activity in the middle of the night. Similarly, recording over 24h the PDF-expressing subset of neurons that control circadian rhythms has revealed a single peak of activity in the evening, which is lost in arrhythmic mutants. The objective of this project is thus to characterize the “spontaneous” activity of the Drosophila brain, with particular efforts on the nocturnal peak of Ca2+-activity that is detected in the MBs. In parallel, we will characterize the neuronal activity of the different subsets of clock neurons, and investigate the interactions between these neurons and the activity of the MBs. Finally, we will study the role of the nocturnal Ca2+-activity in the MBs, in particular its putative function in sleep and/or memory consolidation.
To perform this project, we do have a large number of P[GAL4] lines that allow to drive GA in different brain structures, as well as the particular and unique setup to detect bioluminescence in these conditions. We believe that the detection of neuronal Ca2+-activity, at the spatial resolution of a few microns (possibly at the resolution of a single neuron), in a genetically tractable model system such as Drosophila, represents a real breakthrough. The project will yield to a better understanding of how neural circuitry (neuronal networks) operates and interacts as an ensemble in the nervous system, and likely bring new understanding, at the cellular level, of how neuronal activity is organized to generate behavioural functions such as sleep and/or memory.