Expression of red fluorescing calcium indicators

The pUAST backbone vector includes five upstream activator sequence (UAS) regions as binding partners for GAL4 induced expression [Brand & Perrimon, 1993].

Downstream to the UAS binding site, the R-GECO1.0 DNA was cloned between the NotI and BglII restriction sites. In addition, the mini-white gene was present in the p-element flanked region (Figure 3.26 A).

The p-elements are necessary for the insertion of the enclosed DNA into the Drosophila genome after vector injection in the germline cells [Rubin & Spradling, 1982]. Mini-white is used as a marker for positive germline transformation. The plasmids are injected into flies with a defective white gene and therefore a white eye color. Only flies with an insertion of the transgenic DNA express themini-white gene and have eyes with a red color [Hazelrigg et al., 1984].

0 10 20 30 40 50 60 70 UAS:R-GECO line, expressed in Or83b - Gal4

Rel. fluorescence

5M (II)

Figure 3.27. Evaluation of the expression levels of the UAS:R-GECO1.0 fly lines.

(A)The UAS:R-GECO1.0 flies were crossed with Or83b-GAL4 in order to investigate the expression levels of the dif-ferent lines. After the dissec-tion of the brain, the expres-sion levels of the transgene were tested by measuring the basal fluorescence. The av-erage measured fluorescence from five flies per line differed in the five versions of UAS:R-GECO1.0. The line 5M located on the second chromosome showed the highest basal ex-pression. (B) The functionality of the sensor was confirmed in an imaging experiment in a living fly of line 5M. Pipetting KCl on the brain resulted in a depolarization of the neurons and thereby an increased calcium concentration in the cell which could be detected by the sensor. Scale Bar: 50µm

3. Results

The pCaSpeR:mb247 backbone vector did not contain the mini-white gene but the white-locus to express the white gene for red eye-color in positively transformed flies. Instead of the five UAS regions, the vector included the mb247-promotor for specific expression of the transgene in Kenyon cells of the mushroom body [Riemensperger et al., 2005] (Figure 3.26 B).

During the amplification of the vectors in E. coli, the ampicillin resistance was necessary for the selective amplification of transfected clones. The resistance casette is located outside of the p-elements and therefore not incorporated in the Drosophila genome after injection. The vectors were injected into the germline cells by the company BestGene Inc. (Chino Hills, CA, USA) which also determined the chromosome on which the transgene was incorporated into the genome. After shipment of the flies to Germany, the expression of the transgenes was tested by Ulrike Pech in the laboratory.

The UAS:R-GECO1.0 line was combined with a driver line targeting olfactory sensory neurons, Or83b-GAL4, in order to express the calcium sensor. First, the base line fluorescence without neuronal activation was measured in five different fly strains with UAS:R-GECO insertions. Therefore, five brains per strain were dissected and the basal fluorescence averaged (3.27 A). All five lines expressed R-GECO1.0, but the line 5M with the insertion of the transgene on the second chromosome showed the highest basal fluorescence. This line was used to examine the functionality of the calcium sensor. Therefore, KCl was pipetted on the brain which leads to a depolarization of the neurons. The increase of the fluorescence shown in figure 3.27 B confirmed the functionality of the sensor. The measured signal (^∆F_F

0 ) increased by 150% of the base line fluorescence (F0). Hence, the functionality of the new UAS:R-GECO1.0 fly line could be confirmed.

Similarly, the functionality of mb247:R-GECO1.0 was examined. After determining the line with the highest basal fluorescence, the response to KCl stimulation was observed. As shown in figure 3.28, the signal in the mushroom evoked by KCl application was even stronger than in the antennal lobe. Whereas the UAS line expressed in the olfactory sensory neurons showed an increase in fluorescence of 150% compared to the base line (^∆F_F

0 ), the fluorescent increase in the mb247 line was 350%.

0 10 20 30 40 50 60 70 -50 50 0

100 150 200 250 300 350

400

time (sec)

F/Fo(%)

Figure 3.28. Evaluation of mb247:R-GECO1.0.

Flies expressing the red calcium sensor R-GECO1.0 under the control of the mb247 promotor express the sensor in the mushroom body. Upon stimulation with KCl, the fluorescence of the sensor increases by 350%. The two pictures on the right show the fluorescence of the mushroom body before and during the stimulation with KCl.

Conclusively, it could be shown that the new red fluorescent calcium sensor R-GECO1.0, developed by Zhao et al. [2011] is functional in the fruit fly. The generated fly lines expressing R-GECO1.0 under the control of UAS therefore provide a new tool to investigate neuronal function. With the help of various GAL4-driver lines, the sensor can be expressed in subsets of neurons and used in combination with commonly used green fluorescent proteins to label other structures of interest.

It will even be possible to monitor the neuronal activity in two distinct subsets of neurons when the red sensor is used in combination with a green sensor. For example, the other newly generated line expressing R-GECO1.0 under the control of the mb247 promotor can be used to image neuronal activity in the mushroom body and any subset of neurons targeted by the GAL4 line of choice. Additionally, it will be possible to use UAS:R-GECO1.0 in combination with GCaMP3.0 under the control of another binary expression system, e.g. the LexA/LexOp system [Szüts

& Bienz, 2000; Lai & Lee, 2006]. Therefore, the third newly generated fly line, LexOp:GCaMP3.0, will be of use.

3. Results