In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach

Abstract

Different optical imaging techniques have been developed to study neuronal activity with the goal of deciphering the neural code underlying neurophysiological functions. Because of several constraints inherent in these techniques as well as difficulties interpreting the results, the majority of these studies have been dedicated more to sensory modalities than to the spontaneous activity of the central brain. Recently, a novel bioluminescence approach based on GFP-aequorin (GA) (GFP: Green fluorescent Protein), has been developed, allowing us to functionally record in-vivo neuronal activity. Taking advantage of the particular characteristics of GA, which does not require light excitation, we report that we can record induced and/or the spontaneous Ca(2+)-activity continuously over long periods. Targeting GA to the mushrooms-bodies (MBs), a structure implicated in learning/memory and sleep, we have shown that GA is sensitive enough to detect odor-induced Ca(2+)-activity in Kenyon cells (KCs). It has been possible to reveal two particular peaks of spontaneous activity during overnight recording in the MBs. Other peaks of spontaneous activity have been recorded in flies expressing GA pan-neurally. Similarly, expression in the glial cells has revealed that these cells exhibit a cell-autonomous Ca(2+)-activity. These results demonstrate that bioluminescence imaging is a useful tool for studying Ca(2+)-activity in neuronal and/or glial cells and for functional mapping of the neurophysiological processes in the fly brain. These findings provide a framework for investigating the biological meaning of spontaneous neuronal activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

“…Each component, mathematically deconvolved, had a different contribution of Ca 2+ from intracellular stores and Ca 2+ entry. In a subsequent study , odor‐evoked signals were detected downstream in the olfactory pathway, in the mushroom bodies, confirming the strength of GA bioluminescence for functional brain mapping in vivo . Long‐term, overnight recordings of spontaneous brain activity with high temporal resolution (<10 ms) were also achieved in the mushroom bodies, in glial cells or in all neurons (using a pan‐neuronal promoter).…”

“…In addition to the extensively used synthetic Ca 2+ indicators, a large number of fluorescent and luminescent probes compose the GECI toolbox , each with its own advantages and drawbacks. Ca 2+ ‐sensitive photoproteins are ideally suited for continuous long‐term registration in minimally invasive studies . As drawbacks, exposure times longer than in fluorescence are usually needed because of their low photon yield.…”

A single pair of leucokinin neurons are modulated by feeding state and regulate sleep-metabolism interactions

“…In vivo imaging was performed using a previously described protocol with some modifications [97,98]. Briefly, fed or 24 hr starved flies were anesthetized on ice and secured in 200μL pipette tip with head and proboscis accessible.…”

<em>In Vivo</em> Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

“…Alternatively, depending on the desired experimental conditions, slower acquisition time could be used. For instance, in long O/N recordings, we have used a 2 sec integration times due to the large number of data points collected (Minocci et al, 2013). In brief, one practical feature with the bioluminescence is that the temporal resolution can be easily adjusted according to the desired experimental conditions.…”

“…Odor stimulation has been used to examine activity of brain neurons in previous studies 11,15,16 . Using longer term imaging, however, our lab has been able to show previously unreported changes in kinetics within the olfactory receptor neurons to odors.…”