In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

The recently developed bioluminescent-based GFP-aequorin approach presented here allows in vivo functional recording of Ca²⁺-activity in different neurons, as well as if desired, in other kind of cells such as kidney stellate cells as reported in Cabrero et al., 2013⁵.

Modifications, trouble shooting, additional components, and critical steps

Drosophila Husbandry

GFP-aequorin transgene (pG5A)⁴ was inserted into the pUAST vector to create three independent transformant lines⁶. All lines were tested for their bioluminescence, and all were able to respond to the in vivo Ca²⁺-activity⁶. However more recent experiments have pointed out that the GA2 line inserted on the third chromosome generates more robust signal than the GA1 line inserted on the second chromosome. Here, the representative results were obtained by using UAS-GA2 crossed with the MB-specific line OK107. Moreover, recently B. Pfeiffer has generated a GFP-aequorin construct placed under the control of 20XUAS regulatory element (20X-G5A-2) and this construct has been validated in our lab. Briefly, it gives a very strong signal, stronger than our standard GA2, which therefore should be more efficient and useful to further monitor the Ca²⁺-activity in deeper structures of the brain, such as the ellipsoid-body. Additionally, it may also be useful for imaging small quantities of neurons or in axon terminals (synaptic contacts).

Preparations for Imaging

Although the fly must be ice anesthetized during positioning and gluing, it is important to minimize the time on ice. Flies should be transferred to a glass vial and kept on ice for 2 min before being moved to the chilled Petri dish for positioning in the pipette tip. We have noticed that extended time period of anesthesia reduces the viability of the sample and may attenuate the response. Optimally, we keep the time of ice anesthesia below 5-10 min.

Taping and gluing are critical steps. It is crucial to secure the fly head in place for dissection and imaging as well as to prevent leakage of the Ringer’s solution into the pipet tip and/or out of the recording chamber. Dental glue (and to a lesser extent the silicon glue) will become sticky and subsequently begin to dry as soon as the two components are combined. Therefore it is critical to apply the glue expeditiously to avoid weak bonding and clumping of the glue. In some cases we have noticed that alternate varieties of tape and glue, when in contact with the Ringer’s solution, may release contaminants into the Ringer’s solution which can affect the fly and its response, particularly so for experiments dedicated to the study of olfactory system, using different odors. Consequently, use caution here if substituting materials (see materials for specific varieties we use).

Several different forms of coelenterazine have been developed in order to optimize its activity such as the speed of light emission, Ca²⁺-affinity (sensitivity), or the intensity of light emission¹². For example, the native coelenterazine is more stable, thus more suitable of long term imaging as several hours, while the benzyl-coelenterazine (also named h-coelenterazine) results in a faster and higher light emission with shorter half-life. For additional information about different forms of coelenterazine, see the web address in the Materials List.

Imaging

Bioluminescence signals can be monitored with an electron multiplier CCD camera (EM-CCD, cooled to -80 ºC) fitted onto a microscope. This setup must be housed inside a tight dark box to avoid any undesired (ambient) light contamination. The setup described in this manuscript uses a 20X immersion-objective lens permitting a field of view of 400 x 400 µm (512 x 512 pixels). To improve signal to noise ratio, data were acquired with a 0.100 sec integration time (10 Hz), and 2 x 2 binning was used (1 pixel = 1.2 x 1.2 μm). To acquire and store data, each detected photon was assigned x,y-coordinates and a time point. Between 500 and 600 nm wavelength, the EM-CCD camera (see precise description in Materials List) has a quantum efficiency of 96%.

One of the most crucial parameters for the in vivo functional brain imaging is the temporal resolution. Up to now, the majority of fluorescence-based Ca²⁺-imaging technique such as GCaMP and cameleon provides a temporal resolution of about 4 Hz (250 msec/frame). Recently, with bioluminescence-based GFP-aequorin we have been able to raise the acquisition time up to 100 msec/frame with the EMCCD camera and even up to 120 frames/sec (8.3 msec/frame) with the electron multiplier ICDD camera¹⁰. At this temporal resolution approaches that of the electrophysiological techniques (in the range of about 1 msec). While high temporal resolution is both informative and crucial for the studies dedicated to synaptic transmission and stimulus induced activity, extended slower yet essential Ca²⁺-kinetics also occurs within neurons, for instance the Ca²⁺ release from the intracellular Ca²⁺-stores (for a review: Berridge et al., 2003¹³). For this latter type, a slower temporal resolution is still acceptable, while inversely, they generally require a better Ca²⁺-affinity from the sensor to be detected. This requirement has been achieved with GFP-Aequorin to detect the release of the intracellular Ca²⁺-stores at the axon terminal of the olfactory receptors neurons^7,8. 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.

Alternative chambers (fly’s setup) have been developed to facilitate the objectives of the experiment. For example, for the studies based on natural odor stimulus, the antenna must be kept free, therefore an approach like the one developed by A. Fiala¹⁴ is best suited (we used a similar approach for Murmu et al. 2010). Briefly, in this approach, the fly is tethered (glued) on a minutien pin at the neck, which is glued to a plastic cover slip containing a small hole. A seal is created around the top of the head of the fly. Thereafter, a drop of ringer is deposited on the head, and the head capsule is dissected. In such a way, the antennae are kept free and are available to sense odors. Similarly, for the long term recording, as O/N or even couple of days¹⁰ the fly needs to be kept as free of constraint as possible, and in some cases fed (for example: with a capillary paper soaked in a 5% glucose solution). In these circumstances Fiala’s approach is also more suitable than the approach in the blue pipet tip (snorkeling method).

All drug applications can be controlled externally using a 6 way multi-valves gravity perfusion system. The flow can be controlled using volumetric perfusion regulators calibrated prior to each recording session for a flow of 2 ml/min. Drainage apparatus extracts liquid at a rate of 2 ml/min from the recording chamber via peristaltic pump permitting a continuous flow.

In some conditions, due to expensive cost of the drug and/or the amount of drug to use, one would like to apply only small quantities of compounds. Thus, it is necessary to directly apply it in the bath rather than through perfusion system. In this case the shutter must be closed during this process to avoid the light contamination.

Analysis methods

Counts per sec or counts per sec per coordinate have both been used by our group for analysis. Counts per second is perhaps best suited for bright responses in whole structures whereas counts per sec and per coordinate (which represents a normalization of the responses to the size of the ROI) is best suited and accurate for small populations of cells. Both of them can be easily selected in the analysis software.

One of the main advantages of using the bioluminescence approach is the way that are acquired and stored the data (x,y,t parameters of each emitted photons). Indeed, while any other imaging techniques used standard mode as an acquired full image (generally in a tiff format), with this system, we acquires only the relevant and significant signal by storing only the pixel coordinates which have been activated by the light during a pre-fixed duration of time (which corresponds to the acquisition time or temporal resolution). Consequently, the storage of the data is really “light” compared to the storage a full image, and consequently, makes it easier to analyze the data. For data analysis, we use a related software (photon viewer), which allows setting of the accumulation time as desired. Then the display of an image of the signals can be adjusted as desired in a goal to convincingly present the data. In parallel the results are quantified at the same resolution time than their acquisition time.

Significance and biological applications of bioluminescent imaging

As mentioned above bioluminescent calcium imaging offers three principal advantages to fluorescent calcium imaging techniques: (1) a deeper regions of the brain may be imaged, (2) imaging can occur continuously for longer durations as several hours as O/N and even in some cases up to two days, and (3) more recently, with a higher temporal resolution, up to 120 frame/sec (8.3 msec). The main limitation of the bioluminescent approach is due to its incompatibility with confocal illumination, which consequently does not allow us to determine the precise depth (z-plan) of the activated structures or neurons.

The first advantage of bioluminescent imaging, imaging of deep brain regions, was previously demonstrated in 2007 by the imaging of a high potassium induced calcium response within the ellipsoid body (eb), a deep structure located in the middle of the brain, with no prior electrophysiological or functional reports⁶. Similarly, a more recent example is the recording of the spontaneous Ca²⁺-activity in the eb following application of picrotoxin (a blocker of GABA_A-receptors) demonstrating that the GABAergic ring-neurons of the eb are indeed inhibitory neurons (described in: Diaper et al., article submitted).

The second benefit, longer and continuous time recording, has been exploited in several studies from our lab. 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. The first study by Murmu et al. (2010) showed that while short odor stimuli (1-3 sec) exhibited similar kinetics and a moderate difference in amplitude, a longer odor stimulus (5 sec) caused a much greater amplitude as well as a change in the kinetics/structure of the response which was attributed to intracellular calcium stores. A subsequent study showed that while 5 successive applications of 1 sec odor pulses at 5 min intervals did not change their calcium response, increasing the length of the stimulus to 5 sec led to progressive attenuation of the response, suggesting an adaptation process. Furthermore this adaptation phenomenon appeared to be initiated by GABAergic regulation of previously identified calcium stores⁸. Importantly this data also proves that, in this experimental condition, the GFP-aequorin’s Ca²⁺-sensor can detect and follow the calcium released from the intracellular Ca²⁺-stores. An additional unique study from our lab used bioluminescent imaging O/N to record basal (spontaneous) activity in the whole neuronal and glial populations in the brain as well as activity in the mushroom bodies¹⁰. These recordings revealed surprising spontaneous activity in antennal mechanosensory and motor center (AMMC) and antennal lobe. Furthermore, unexpected cell autonomous activity in the glia occurring throughout the night. Within the mushroom body the study identified both punctate activity and two distinctive peaks, one short and one long whose incidence changed with age¹⁰.

In the most recent study in our lab, we used bioluminescent imaging to examine the effects of the manipulation of secondary signaling molecules implicated in memory, and how they affect the calcium response within the mushroom bodies⁹. Indeed, although dunce and rutabaga were identified more than 30 years ago and are known to regulate the level of cAMP, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca²⁺-response still remain largely unknown. With the GFP-aequorin approach, we have been able to simultaneously record both the calyx (the dendritic structures) and the cell-bodies, as well as the axonal projections at the level of the lobes, and thus allowing us to correlate the level and the kinetic of the response in the different compartments of this highly complex structure.

The most recent application we are developing mimics a natural response through artificially activating neuronal populations presynaptic to the structure of interest. To do this we express the P2X₂ receptor, a ligand-gated cation channel which is responsive to ATP, in the presynaptic neuron and express GFP-aequorin in the post-synaptic neuronal population. The segregation of these two elements into different neuronal populations requires two separate expression systems. To this end a LexA construct containing GFP-aequorin has been created. In our preliminary experiments with this system we have expressed P2X₂ in a subset of the PNs using the Gal4-GH146 with UAS-P2X₂ and GFP-aequorin in the mushroom body using the LexA MB247 line combined with LexA-13X-GFP-aequorin. We have successfully recorded calcium responses in the mushroom body with stimulations of 1 mM ATP to the P2X₂ receptor in the PNs (Figure 6).

In conclusion, the bioluminescence GFP-aequorin approach has proven to be useful in recording the stimulus induced Ca²⁺-activity, analogous to other Ca²⁺-indicators. But more importantly, it also permits the detection of spontaneous Ca²⁺-activity within the brain. This approach opens up future possibilities for long term experiments and whole brain imaging that can increase the depth of our understanding of the physiological phenomena and activity patterns within the brain.