Abstract
Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI’s uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.
Highlights
Calcium is an essential secondary messenger in neurons and has proven to be one of the most widely used signals to trace neural activity (Palmer and Tsien, 2006)
The signal of photoconverted Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) is present for 5% AA, but not when the acetic acid has been neutralized
Maximum intensity projection (MIP) of a single antennal lobe, as designated by the white dotted box in Figures 2C,D, are shown for both 5% AA and neutralized AA. These results confirm that photoconversion of CaMPARI in intact, freely-moving adult flies exposed to acetic acid is specific to the DC4 glomerulus
Summary
Calcium is an essential secondary messenger in neurons and has proven to be one of the most widely used signals to trace neural activity (Palmer and Tsien, 2006). This involves the use of standard Petri dishes with passive humidification and a series of 405 nm LEDs that allow for photoconversion of CaMPARI in the freely-moving intact adult fly With this setup, we demonstrate that photoconversion of specific neuronal circuits is possible in multiple sets of fruit flies simultaneously. We provide quantified photoconversion of an olfactory glomerulus known to be activated by exposure to acids (Ai et al, 2010) as a proof of principle that this method allows for quantifiable significant photoconversion through the intact head cuticle of the freely-moving adult fly Because this method is scalable to any group size, we suggest that CaMPARI in Drosophila is adaptable for large scale genetic screens and/or discovery and mapping of novel circuits that are activated by the investigator’s stimulus of choice. Microscopy Sciences 63434-06 Lot# 171020). 20 × 50 mm No 1 1/2 Coverslips (VWR 48393 194)
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