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Bioluminescence at the service of a novel cerebral imaging technique

CNRS scientists in collaboration have developed a new technique for the in vivo imaging of neuronal function using bioluminescence, based on a GFP-aequorin fusion protein. This imaging technique enables the monitoring of neuronal activity (and more specifically, calcium activity), real-time and in-vivo, in either a small group of neurons or in the brain as a whole.

Participating in the development were The Molecular Embryology Unit (CNRS/Institut Pasteur) in collaboration with the Cellular and Molecular Neurobiology Laboratory (CNRS) and the Neurobiology Laboratory for Learning, Memory and Communication (CNRS/Université Paris-Sud).

The novel imaging technique employs a new, GFP-aequorin marker/tracer. This is a calcium-sensitive protein, which in the presence of its co-factor , coelenterazine, will emit light (a photon) when there is a change to the calcium concentration in a cell; for example, following neuronal activation. This makes it possible to follow neuronal activity in neurons, or even to trace it in a network of neurons. Furthermore, this little-invasive and non-toxic approach allows the recording of neuronal activity over periods of several hours. It is thus possible to monitor the cerebral activity of a Drosophila fruit fly for 24 or even 48 hours.

Because of these characteristics, the new tracer can demonstrate new physiological phenomena related to calcium activity. Thus the activation by nicotine of pedunculate bodies (an important structure for learning and olfactory memory in the Drosophila) induces a secondary response which is delayed by about 10 to 15 minutes at the level of neuronal axonal projections. It is therefore probable that this new response (hitherto totally unsuspected) intervenes in learning and memory phenomena.

Furthermore, using this imaging technique, it has been possible to record neurons in the ellipsoid body, a structure involved in regulating locomotor activity. This structure is deeply embedded in the centre of the brain and has never been studied physiologically because it was always inaccessible to standard, fluorescent-type markers. Such recordings of the ellipsoid body have thus demonstrated the considerable sensitivity of this novel approach, while validating access to all structures, even those located deep in the brain. It will therefore be possible to study all neurons or cerebral structures using this approach.

This novel technique opens numerous perspectives. This tracer can thus be expressed in all cells of the brain nervous system (both neurons and glial cells) in order to monitor the activity of the whole brain. Preliminary data are already available. For the first time, it is possible to draw up anatomical and functional maps of the brain (in this case, of the Drosophila) based on long-term recordings. Such maps do not yet exist for any animal species, including man.

A funding application has been made to the ANR in order to build up functional maps of the Drosophila brain. These maps are a prerequisite to producing a mass of data on the global activity of the entire brain which will then serve as a reference to compare cerebral activity in different contexts: for example, differences between males and females (demonstration of sexual dimorphism) or as a function of age (changes to cerebral activity during ageing).

The Drosophila is an excellent model for the study of ageing and longevity, because it has recently been shown that flies endowed with a mutation of the insulin receptor live much longer. It is also possible to exploit the powerful genetic tools of the Drosphila to study and compare these maps in flies bearing different mutations, or in flies which serve as models for a variety of human pathologies, such as Alzheimer's disease, Parkinson's disease or Huntingdon's chorea, or in the context of a pharmacological approach regarding addiction to different drugs (alcohol, ni cotine, cocaine, etc.).


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