Sensitivity of the receptor is unaltered by the reference odor used

In order to see if the reference odor could have an impact on the half maximal response of the odorants, we tested dose responses of 6 odorants (10^-2 to 10^-6 dilutions;

Fig 5.11). Four of the odorants selected, elicited significant excitatory responses (LINT, TerT, EugM and CaRT) and two of them elicited inhibitory or insignificant responses (FurL, BBtL). BBtL elicited inhibitory responses when IPES was used as a reference odor, whereas it elicited weak responses when MBAE was used as reference odor at 10^-2 dilution (these responses were insignificant). Excitatory odorants elicited significant response to 10^-2 and 10^-3 dilution alone (Fig 5.11), whereas inhibitory odorants elicited insignificant response across concentrations (Fig 5.11) as observed before. Dose response curves were similar as before (for example curves see Fig 5.12) and the hill coefficient and EC₅₀ values were estimated from the curves as described before. For all odorants hill coefficients were greater than 1, but the values are lower than the values obtained before, but still these values are higher and correspond to the requirement of high odor concentration. The EC₅₀ values spanned about 2.8 – 3.4 log units (Fig 5.12 and Table 5.4). Sensitivity of most odorants was unaltered except for EugM and TerT (for TerT the shift is insignificant because of low n).

Chapter 5. Results

124

Figure 5.12. Dose response curves (mean ± s.e.m., red colored trace) overlaid with fitted Hill curves (black colored trace) for four of the excitatory odorants tested . Reference odor used is MBAE, n=3 – 5 flies.

Chapter 5. Results

125

Table 5.4. Sensitivity of Or69a ORNs to the excitatory odorants tested, reference odor – MBAE

Odorants Fixed Hill co-efficient used EC50

values between individual odor presentations and odor blocks, the mineral oil response was still contaminated with the odor (IPES) response. Thus it seems that this odorant is very sticky and cannot be washed away completely from the syringe by the method used for other odorants. However, the percentage of odorants that elicited statistically significant responses and response dynamics of Or69a neurons were altered less by IPES. About 15% of odor responses (5 out of 32 odorants tested) were altered (Figs 5.2, 5.9 and Table 5.3) and were majorly independent of the similarities in chemical moieties.

Nonanoic acid and IPES belonged to the same chemical group, whereas other odorants were chemically dissimilar. Odorants that elicited strongest responses (best ligands) was shifted when the reference odor was changed. This shift can be omitted, because we also observed similar effect when the concentration of the odorant was changed (best ligand differed between 10^-2 and 10^-3 dilution even when same reference odor was used (IPES);

Figs 5.2, 5.4). All of these results indicate that the response profile and sensitivity of Or69a remained similar regardless of the reference odor used (IPES or MBAE). From all our results we could conclude that Or69a is a broadly tuned receptor, since about 40%

of the odorants we tested, activated the receptor. Almost all of these odorants elicited excitatory responses. Only FurL elicited significant inhibitory responses. Higher concentrations of odor molecules were required for half maximal activation of the receptor (EC₅₀ values spanned around 2.8 – 3.8 log units). Best ligands of the receptor belong to the group of terpenes (Tert, LINT, CaST and CiLT), esters (E3HE) and aromatics (EugM).

Chapter 5. Discussion

126

5.5 Discussion

The combinatorial nature of olfaction allows the brain to recognize and remember thousands of different odors with a limited number of olfactory receptor types. In order to understand how the brain perceives an odor, the response profiles of all receptors for a given species should be known. The response profile of an ORN is not only predicted by the OR it expresses. Rather, it also depends on the OBPs (Pophof, 2004; Xu et al., 2005), on the ORNs which are housed in the same sensillum (Dobritsa et al., 2003), on co-receptors (Benton et al., 2006; Dobritsa et al., 2003; Larsson et al., 2004;

Neuhaus et al., 2005) and G proteins (Chakraborty et al., 2009; Deng et al., 2011; Kain et al., 2008; Shirokova et al., 2005; Wicher et al., 2008). Thus, the response profile of a receptor, which is characterized within an intact animal, will give a better picture than in vitro predictions. In this study we characterized the response profile of the ORNs expressing the receptor Or69a by in vivo calcium imaging of the antenna.

Intracellular calcium changes are used to monitor the cellular activity. Calcium in dendrites is correlated with the elicited receptor potential and might be a combination of extracellular calcium entering through voltage or G protein gated calcium channels and the calcium released from intracellular stores via G protein activated channels (e.g. IP₃ receptors in endoplasmic reticulum (ER)) or calcium sensitive channels (e.g. Ryanodine receptors in ER) or through mitochondrial calcium mobilization (Fluegge et al., 2012).

The dendritic calcium change is shown to be involved in signal transduction and adaptation in vertebrates and insects (Matthews and Reisert, 2003; Stengl, 1994). In invertebrate sensory neurons cell bodies were shown to elicit a delayed calcium response for stimulation (Hoger et al., 2005). We quantified the calcium changes in the dendritic and somatic compartment of Or69a neurons and it may correlate to the spike activity of the neuron as reported previously for other receptors (Pelz et al., 2006). Our results show that Or69a is a broadly tuned receptor, with a majority of activating odorants and a few inactivating odorants (Figs 5.2, 5.4, 5.9).

In our screen majority of the odorants activated the receptor and few odorants inhibited the receptor. Or69a was activated by many odorants (for e.g. LINT, TerT, CaST, EugM, CaRT) and inhibited by few odorants (e.g. FurL, BBtL, OctS, HexS). The identity of a stimulus at a particular concentration in the natural environment can be encoded not only by the identity of the responding receptors but also by the temporal dynamics of their responses (Martelli et al., 2013; Raman et al., 2010). Odor evoked spike trains in Drosophila ORNs were shown to be odor and receptor specific (de Bruyne et al., 1999; de Bruyne et al., 2001; Nagel and Wilson, 2011; Schuckel et al., 2009). Odorants that activated Or69a elicited different dynamics irrespective of their chemical properties and intensities (Figs 5.4, 5.6, 5.7, 5.9). This could help the animal to code for identity of the odor. Inhibition of ORs by odorants; odor induced reduction of basal ORN activity,

Chapter 5. Discussion

127

has been documented in both vertebrates and invertebrates (Reisert and Restrepo, 2009) apart from activation. In Drosophila it has been shown that an individual odor can activate some receptors and inhibit others and also a receptor can be activated by some odorants and inhibited by others. The existence of two response modes may add a degree of freedom to odor coding (de Brito Sanchez and Kaissling, 2005). Though Or69a was inhibited by many odorants only FurL elicited statistically significant inhibition. FurL was shown to activate (e.g. Or7a, Or85b, Or67a and so) and inhibit few odorant receptors (e.g. Or13a, Or47b, Or67b and so) of Drosophila (refer DoOR database). In this study we report that FurL also inhibits the receptor Or69a. It should be noted that FurL (at 10^-2 or 10^-3 dilution) elicited a complex response, after odor stimulation we observed a short increase in calcium response and then a decrease in calcium response (Figs 5.3, 5.7, 5.10). I speculate that may be this odor could activate different transduction cascades and thus lead to the complex response observed.

Though many odorants activated the receptor the dose response of the receptor (Figs 5.5, 5.6, 5.11, 5.12; EC₅₀ values were in the range of 10^-2.8 to 10^-3.8) indicate that Or69a is a less sensitive receptor or we may not have found the best ligand for the receptor, yet. ORNs may be specialists which respond to a few key substances or generalist that responds to large set of odorants (Hildebrand and Shepherd, 1997) or both specialists and generalists (can respond to large set of odorants, but few odorants can be detected even at very low concentrations) (Ha and Smith, 2006; Pelz et al., 2006).

For example ORNs expressing Or92a, Or22a and Or67d can be referred to as generalist, generalist and specialist, specialist respectively (de Bruyne et al., 2001; Ha and Smith, 2006; Pelz et al., 2006). Or67d neurons respond only to the sex pheromone (cVA) and can detect the odorant even at very low concentrations (less than 1 %) (Ha and Smith, 2006). Or22a neurons respond to many general odorants, but are highly sensitive to few odorants, specialist for these odorants, half maximal response of ethyl hexanoate and methyl hexanoate is ~ log 10^-7 dilution (Pelz et al., 2006). ORNs expressing Or69a can be termed as generalist, since ~38 odorants out of 105 odorants tested, gave statistically significant responses at 10^-2 dilution.

Odorants that strongly activated the receptor, Or69a (e.g. LINT, CiLT, CaST, EugM, Oc3L, E3HE) are present in many natural sources. For example LINT is present in many flowers and spice plants and is used as an insecticide (for flea and cockroach) also used in some mosquito repellent products. CiLT is found in citronella and rose oils and used as an insect repellent (Taylor and Schreck, 1985). CaST is present in most essential oils and is abundant in the oils of caraway and dill and has been proposed to be used as a mosquito repellent. EugM is present in many spicy plants like cinnamon, bay leaves, basil and is used as insect attractants. It is one of many compounds that are attractive to males of various species of orchid bees (Schiestl and Roubik, 2003) and also

Chapter 5. Discussion

128

attracts female cucumber beetles. E3HE and Oc3L are found in fruits like mango and passion fruit (Pelz et al., 2006). Odorants that strongly activated Or69a are ecologically relevant. Few of the odorants that strongly activated the receptor are repellents for some insect species; this may or may not be true for flies and other odorants that strongly activated the receptor are present in fruits and these odorants may act as an attractant for Drosophila (“fruit flies”). I speculate that Drosophila may use Or69a receptor to sense fruity and general (dangerous chemicals) odorants in the environment. Or69a detects a broad range of chemicals, but requires high concentrations of odor molecules for activation.

This is the first study that reports the response profile for Or69a neurons. This new data set will be integrated into DoOR and fills a gap in the database.

129

Conclusions and Outlook

The main objective of this work is to study the role of G proteins in olfactory signaling of insects. Drosophila odorant receptors, Or22a and Or92a were used as the model for insect ORs and the role of G proteins (G_o/i subgroup of G proteins or G_o and G_q proteins) for olfactory signaling was studied by combined in vivo and in vitro approach.

The results indicate that different ORs activate a G protein to a varying degree and an OR can activate more than one G protein upon odor detection. Also it should be noted that G protein mutations reduced or enhanced the odor response but didn’t abolished the odor response. Two possibilities may explain the results observed, reduction of these proteins was not complete or these proteins indicate only a part of the transduction cascade; other G protein mediated pathways than the G protein tested in this study may play a role or a G protein independent pathway (ionotropic pathway) may contribute to the odor response. From all these results we conclude that olfactory signaling in Drosophila (insects) is complex and we propose that insects use multiple pathways for olfactory signaling, and activate diverse G proteins upon odor detection.

I developed an in vitro olfactory receptor assay for insect ORs, as a tool to study a role of G proteins in olfactory signaling. This assay was used to study a role of G_o/i subgroup of G proteins in olfactory signaling of Or22a neurons. In this assay the percentage of cells that responded to the odor was much higher than reported in the literature (Kiely et al., 2007; Neuhaus et al., 2005; Smart et al., 2008). As a next step we can use this assay to study the role of G_o and G_q in olfactory signaling of the odorant receptor 92a. Also we can use this assay to validate a part of the putative model proposed in Chapter 4. In the model we proposed that GIRKs may represent a possible target protein that is modulated by G_o and G_q. In order to test this hypothesis we can co-express GIRKs together dORs and the odor mediated calcium changes in these cells can be measured and it can give insight on the role of G_o and G_q. By using this assay one can also characterize a role of G proteins in pheromone transduction (cVA) of Drosophila (a model for insects) and for this purpose the gene encoding for this receptor was cloned in mammalian expression vector pCDNA3.1(+). Also this assay can be used by others to de-orphan the ORs from different insect species. The mechanisms of odor adaptation known to date are mostly from vertebrate ORNs and very little is known in this regard for insect ORNs. Though this assay may not reveal all the mechanisms of odor adaptation as it is only suitable to those linked to ORs, yet it can give useful insights about odor adaption in insects.