Odor mediated calcium changes in the antenna (dendro-somatic compartments) were measured by monitoring the genetically encoded calcium dependent fluorescent sensor G-CaMP1.3 in Or92a ORNs. Calcium responses were quantified from the fluorescence emitted through the intact cuticle from an area (as shown in Fig. 4.1A) corresponding to the area of expression of Or92a (Fishilevich and Vosshall, 2005).
Responses increased with odor concentration. For 2, butanediol (BDOL), 2, 3-butanedione (BEDN) and 3-hexanone (3-HXN) they were in the range of 0.5% to 3% of
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We inhibited Go by co-expressing PTX, and reduced the levels of Gq by driving an RNA-interference construct. The efficiency of both transgenic lines in affecting Go and Gq has been tested before (Kalidas and Smith, 2002; Katanaev et al., 2005). Calcium responses in the dendrites of sensory cells do not only reflect signal transduction cascades, but also events linked to sensory adaptation (Leinders-Zufall et al., 1998). Since these are also linked to second messenger cascades we specifically addressed whether Gq or Go are involved in sensory adaptation or OR signal transduction by choosing double pulse protocol (two odor pulses of 1 s each with an inter-stimulus interval of 2 s) for odor stimulation. Responses to the first odor pulse can be used to quantify the role of G proteins in signal transduction cascades (would expect a decrease in calcium fluorescence compared to control) and response to second pulse can be used to quantify the role of G proteins in adaptation (would expect a increase in calcium fluorescence compared to control) and or in signal transduction cascades (like the response to the first odor pulse).
Time window used for the calculation of first and second odor pulse are shown in Fig 4.1B.
In general adaptation was observed in all flies including wild type types in that the response of second odor pulse was lower than first odor pulse. Odor responses in mutant flies with disturbed G alpha subunits markedly differed from those of wild types (Fig 4.1E). The calcium response of the 1st and 2nd odor pulse was affected to the same extend in all treatment groups (mutant genotypes: Go, Gq, Go and Gq). Hence we quantified the response of 1st and 2nd odor pulse together (Fig 4.1C) and the results shown further were calculated accordingly. The nature of observed effects differed between odorants, concentration as well as sex of the flies.
Flies with mutated G alpha subunits show enhanced odor responses as compared to wild type flies.
Measurements on the antenna show that reduced levels of Gαo in Or92a neurons lead to increased odor responses as compared to control flies (Fig 4.2). This effect is observed regardless of odor identity and fly’s sex. At highest concentrations reduced levels of Gαo lead to a significant increase in odor response (p < 2e-16) as compared to the control flies for all odorants and both males and females (Fig 4.2A-D). At low
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concentrations Gαo mutants elicited significantly higher responses than control flies only for some of the odorants tested (Fig 4.2E- J). Reduced levels of Gαo in all the ORNs expressing the co-receptor (Orco-GAL4) were shown to reduce (Chatterjee et al., 2009) or unalter (Deng et al., 2011; Yao and Carlson, 2010) the odor induced spike activity or EAG amplitudes, regardless of odor identity and intensity in Drosophila ORNs. But we found that when the levels of Gαo is reduced in Or92a neurons, odor responses were increased for most odorants and concentrations for Or92a neurons, it should be noted that we used a OR-GAL4 (expression in a specific ORN) to drive the expression of RNAi construct specific for Gq, whereas other studies used Orco-GAL4 (Deng et al., 2011; Yao and Carlson, 2010).
Like Gαo mutants, Gαq mutants also elicited higher odor responses when compared to control flies except for BEDN at 10-2 dilution in male flies (data from MT), irrespective of sex and odor identity, but not for odor intensity (Fig 4.3 A-D). At some concentrations Gαq mutants elicited insignificant response when compared to control. In some cases an odor at a particular concentration elicited different response in males and females. For example the odor BDEN at 10-2 dilution elicited higher response in Gαq mutants than control females, whereas it elicited lower or insignificant response in males (Fig 4.3B). Mutations in the Drosophila dgq-gene encoding the Gq subunit, were shown to reduce odor responses across concentrations in D. melanogaster (in vivo; SSR) (Kain et al., 2008), whereas reduced or higher levels of Gq was shown to unalter the odor response (Deng et al., 2011; Yao and Carlson, 2010). But we found an enhanced response to odors upon inhibition of Gq in Or92a neurons, but it should be noted that we used a OR-GAL4 (expression in a specific ORN) to drive the expression of RNAi construct specific for Gq, whereas other studies used Orco-GAL4 (Deng et al., 2011;
Yao and Carlson, 2010) or PAN-GAL4 (Kain et al., 2008) driver (expression in ORNs as well as in the supporting cells in a sensilla) for Gq downregulation.
Reduced levels of both Gαo and Gαq (double mutant)in Or92a neurons elicited higher response than control flies for most odorants at high concentrations (except for BEDN in males; double mutant elicited lower response than control) like Gαo or Gαq mutants (Fig 4.4A-D). At low odor concentrations some odorants elicited higher response in double mutants than control, whereas others elicited lower response in double mutants than control (Fig 4.4E-J). As observed in Gαq mutants for some odorants at a particular concentration, odor response differed in males and females (Fig 4.4E, G, I, J).
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Gαo mutants show enhanced odor responses as compared to Gαq mutants at high odor concentrations
In general Gαo mutants elicited higher response than Gαq mutants at higher concentrations independent of sex and largely independent of odorant (Fig 4.5A- D).
An exception from this general trend is observed when stimulating with the odor ESHE.
For this odor Gαq mutants elicited higher responses than Gαo mutants (Fig 4.5D). On the contrary, at low concentrations female Gαo mutants elicited higher response than female Gαq mutants. For males this effect was reversed (Fig 4.5E-J).
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Figure 4.1. Antennalcalcium imaging of Or92a ORNs; Go and Gq modulates odor response.
(A) Morphological view of an antenna of a male Drosophila melanogaster, overlaid with the false color coded picture of response to 2, 3 butanediol (BDOL) 10-2 dilution. Black dotted lines mark the margin of the antenna. Image was taken from a CCD camera. Black circle indicate the area from which responses were calculated. (B, C) Mean traces (shades indicate s.e.m.) of response to the reference odor, ethyl-(S)-3-hydroxybutyrate (ESHE) at 10-2 dilution for the double pulse odor stimulation protocol. Data points for first 5 s of stimulation are not shown. Gray bars in the plot indicate the time and duration of odor delivery.
Closed and dotted green square in the trace (panel B) indicate the time window (ΔF/F value of every 250 ms (every frame) was used not the average or peak response) used for the statistical comparisons of the odor response for 1st and 2nd odor pulse respectively. Black square in the trace indicates the time frame used for the quantification of odor response (ΔF/F value of each frame; panel C). (D) Mean traces (shades indicate s.e.m.) for 3-hexanone (3HXN; left), BDOL (middle) and 2, 3 butanedione (BEDN; right) to the concentrations 10-6 (light red), 10-4 (red) and 10-2 (dark red) dilution) tested in control males. Asterisks indicate statistical significance for all the concentrations tested, two way ANOVA (concentration and frames (time scale indicated in panel C) are used as factors; significance codes; ‘***’ 0.001). (E) Mean traces (shades indicate s.e.m.) for 3HXN (left), BDOL (middle) and BEDN (right) to all genotypes tested to 10-2 dilution. Red, blue, green and cyan colored traces indicate control, PTX (reduction of Gαo), Gαq
RNAi (downregulation of Gαq) and PTX and Gαq RNAi (double mutant) group of flies respectively. n = 4 – 7 flies for every genotype, male data JI.
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Figure 4.2. Odor mediated calcium changes in the antennae of D. melanogaster are enhanced by the levels of Gαo.
Mean traces (shades indicate s.e.m.) for BDOL (10-2 (panel A), 10-4 (panel E) and 10-6 (panel H) dilution), BEDN (10-2 (panel B), 10-4 (panel F) and 10-6 (panel I) dilution), ESHE (reference odor; 10-2 dilution (panel D)) and 3HXN (10-2 (panel C), 10-4 (panel G) and 10-6 (panel J) dilution) for males (left (data from JI) and middle (data from MT) panels) and females (right panels) to few genotypes tested. Red and blue colored traces indicate control and PTX (reduction of Gαo) group of flies respectively. Asterisks indicate statistical significance, two way ANOVA (treatment and frames (time scale indicated in Fig 4.1C) are used as factors; significance codes; ‘***’ 0.001). n = 5 – 8 flies for every genotype.
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Figure 4.3. Odor mediated calcium changes in the antennae of D. melanogaster are enhanced by the levels of Gαq.
Similar to figure 4.2, but the genotypes tested were control and Gαq RNAi (downregulation of Gαq) group of flies respectively. Red and blue colored traces indicate control and Gαq RNAi group of flies respectively.
Response of mutants differed for few odorants at particular concentrations; those traces were labeled with grey square. n = 5 – 8 flies for every genotype.
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Figure 4.4. Odor mediated calcium changes in the antennae of D. melanogaster are enhanced by the levels of Gαo and Gαq.
Similar to figure 4.2, but the genotypes tested were control and PTX and Gαq RNAi (double mutant) group of flies respectively. Red and cyan colored traces indicate control and PTX and Gαq RNAi group of flies respectively. Response of mutants differed for few odorants at particular concentrations; those traces were labeled with grey square. n = 5 – 8 flies for every genotype.
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Figure 4.5. Gαo mutants exhibits higher response than Gαq mutants at high odor concentrations and are vice versa at low concentrations.
Similar to figure 4.2, but the genotypes tested were PTX (reduced levels of Gαo) and Gαq RNAi (downregulation of Gαq) group of flies respectively. Blue and green colored traces indicate PTX and Gαq
RNAi group of flies respectively. Response of mutants differed for few odorants at high or low concentrations and was labeled with grey squares or grey circle respectively. n = 5 – 8 flies for every genotype
Male double mutant show lower odor responses than single mutants
In general Gαo and Gαq mutant males (double mutant) exhibited lower odor responses than the single mutant males (Gαo or Gαq). This difference was significant (p <
2e-16) for a majority of the odorants. Some odorants (BEDN and 3HXN at 10-6 dilution) elicited at low concentration significantly higher responses indouble mutants than in single mutants (Fig 4.6B, C). In general double mutants did not show enhanced odor responses than single mutants, but rather a decrease in response was observed.
Female double mutants show higher odor responses than single mutants for high odor concentrations.
Responses of female flies differed from those of males only at high concentrations (10-2 dilution). At the highest concentration that was tested, all odorants, except ESHE, elicited higher responses in female double mutants than in female single mutants (Fig 4.7C). At low concentrations (10-4 and (or) or 10-6 dilution) double mutants show lower responses than single mutants for most odorants, which is in well accordance with our findings for male flies (Fig 4.7A, B). Overall these results indicate that at low odor concentrations Gαo or Gαq mutants antagonize the effect of other, whereas at high odor concentrations Gαo or Gαq enhances the effect of other.
Taken together reduced levels of Go or Gq or both Go and Gq enhance the odor response as compared to control flies. Antennal imaging data of JI and MT are comparable, which indicates that the effect of G protein mutants on odor response is reproducible. We conclude that both Gαo and Gαq modulate odor responses in the dendro-somatic compartment of Or92a ORNs. Both proteins enhance the odor response when they are activated alone, whereas co-activation of these proteins antagonize each other and the response is unmodulated; like the response of control flies.
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Figure 4.6. Double mutant elicited lower response than the respective single mutants in males.
(A, B, C, D) Mean traces (shades indicate s.e.m.) for BDOL, BEDN, 3HXN and ESHE respectively to all concentrations (respective concentrations are mentioned above the traces) and genotypes tested in males.
Blue, green and cyan colored traces indicate PTX, Gαq RNAi, and PTX and Gαq RNAi group of flies respectively. Top and bottom rows of every panel represent the data from JI and MT respectively.
Response of PTX flies differed (compared to double mutant) for few odorants at particular concentrations; those traces are labeled with grey square with blue asterisk. Asterisks indicate statistical significance compared to double mutant, two way ANOVA (treatment and frames (time points are indicated in Fig 4.1C) are used as factors; significance codes; ‘***’ 0.001). n = 5 – 8 flies for every genotype.
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Figure 4.7. Double mutant elicited lower response than the respective single mutants at low odor concentrations alone in females.
(A, B, C) Mean traces (shades indicate s.e.m.) for 10-6, 10-4 and 10-2 dilutions respectively for all genotypes tested in females. 1st, 2nd, 3rd and 4th rows of each panel indicate the odorants tested; BDOL, BEDN, 3HXN and ESHE. Blue, green and cyan colored traces indicate PTX, Gαq RNAi, and PTX and Gαq RNAi group of flies respectively. Response of single mutants (PTX or Gαq RNAi or both) differed (compared to double mutant) for most odorants at high concentrations than males and are labeled with magenta colored square. Asterisks indicate statistical significance compared to double mutant, two way ANOVA (treatment and frames (time points are indicated in Fig 4.1C) are used as factors; significance codes; ‘***’ 0.001). n = 5 – 8 flies for every genotype.
4.4.3 Calcium imaging of odor responses in the antennal lobe (axon terminals)