Ca 2+ -imaging of the three used odorants in the antennal lobeantennal lobe

The structural similarity of 1-Oct and 3-Oct was confirmed on a behavioral level as a strong generalization could be observed. To investigate whether this similarity can also be found on a physiological level, the neuronal activation evoked by the three odorants used during the behavioral experiments (1-Oct, 3-Oct and MCH) was measured with Ca²⁺-imaging in the antennal lobe. By the means of calcium indicators, the intracellular Ca²⁺-concentrations can be monitored. The changes in Ca²⁺-dynamics are a measure for neuronal activity as voltage depended Ca²⁺-channels are opening upon depolarization of the neurons [Stallcup, 1979;

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Figure 3.8. Ca²⁺-dynamics measured in the antennal lobe - focal plane 1.

(A) Example image of the first focal plane with outlines of identified glomeruli. In-creased calcium activity in response to odorant application is depicted in false col-ors, superimposed on the image taken before stimulus onset. Signals lower than 50%

of the maximum signal are cut off. In this focal plane, no response to MCH can be observed. The DM2 glomerulus responds to 3-Oct and 1-Oct. (B) Average intensity changes measured in 7 flies over time in the DM2 glomerulus (± SEM). A weak re-sponse to MCH can be observed, whereas the intensity increase during 1-Oct and

3-Oct application is more prominent. Scale bar: 10µm

Miyawaki et al., 1997; Berridge, 1998]. The spatial patterns of neuronal activity elicited by an odor are describing the odor representations within the structure of interest [Fiala et al., 2002; Griesbeck, 2004; Knöpfel et al., 2006].

The Or83b-Gal4 driver line [Wang et al., 2003a; Larsson et al., 2004] was used to express the genetically encoded calcium sensor GCaMP3.0 (under UAS control [Tian et al., 2009]) specifically in olfactory sensory neurons. Each of the odorants was presented in the same dilutions as during the behavioral experiments and transported with an air stream directly to the antennae of the fly. Intensity changes of the fluorescence reflect an increase in calcium concentration during the appli-cation of an odorant and are therefore a measure of neuronal activity [Guerrero

& Isacoff, 2001; Griesbeck, 2004]. Intensity changes were normalized to the baseline response and plotted over time for selected glomeruli. The glomeruli were identified with the help of optical slices and a 3D reconstruction of the antennal lobe [Laissue et al., 1999] available at http://www.flybrain.org [Armstrong et al., 1995].

Odorant-evoked changes in Ca²⁺-concentrations in the antennal lobe were imaged in two different focal planes in order to get a signal from all three odorants and a measurement for odor similarity. The false-color coded images shown in figures 3.8 and 3.9 visualize areas with an intensity increase of at least 50% of the maximum signal.

The first focal plane is located in the dorsal part of the antennal lobe and includes the DM2 glomerulus innervated by Or22a and Or22b neurons [Couto et al., 2005].

DM2 responded with a strong increase in the intracellular calcium concentration to the application of 3-Oct and 1-Oct but showed no signal towards MCH (Figure 3.8 A). Besides the response in DM2, no odorant evoked responses to MCH, 1-Oct and 3-Oct could be observed in this focal plane. In figure 3.8 B the change of fluorescence intensity and therefore the increase in calcium concentration as a measure of neuronal activity over time is depicted. Whereas the solvent mineral oil did not elicit any response, the glomerulus responded with a weak intensity increase towards MCH. However, the responses during 1-Oct and 3-Oct application were considerably higher. This result is in line with previous studies about olfactory receptor and their ligands including Or22 and 3-Oct and/or 1-Oct [Galizia et al., 2010; Hallem et al., 2004; Fishilevich & Vosshall, 2005; Hallem & Carlson, 2006;

Schmuker & Schneider, 2007].

3. Results

Figure 3.9. Ca²⁺-dynamics measured in the antennal lobe - focal plane 2.

(A)Outlines of identified glomeruli on an example image of the second focal plane. Su-perimposed on the image taken before the stimulus are false-color coded increased calcium concentrations in response to odorant applications. Signals lower than 50%

of the maximum signal are cut off.(B-D) Average responses of 7 flies upon presen-tation of the three odorants over time in the three glomeruli with an intensity change of more than 50% of the maximum signal (±SEM). VC3 responded weakly to MCH and strongly to 1-Oct and 3-Oct. In contrast, only MCH elicited a response in VA3. In VL2 only the presentation of 1-Oct resulted in an increase of fluorescence, whereas no response could be observed towards 3-Oct and MCH. Scale bar: 10µm

The second focal plane is located at the ventral side of the antennal lobe and includes the VC3, VA3 and VL2 glomeruli (Figure 3.9 A). VC3 is innervated by neurons expressing Or35a, whereas VA3 consists of Or67b positive neurons [Couto et al., 2005]. The olfactory receptor expressed by cells innervating VL2 could not be identified, even though a response to alcohols is described [Rodrigues, 1988] and an innervation by antennal coeloconic receptors is proposed [Couto et al., 2005].

VC3 responded weakly to a presentation of MCH and strong to 1-Oct and 3-Oct. A response to the solvent could not be observed (Figure 3.9 B). VA3 on the other hand responded only to MCH and did not show any change in the intracellular calcium concentration in response to 1-Oct and 3-Oct presentation (Figure 3.9 C). The responses obtained in VC3 and VA3 are comparable with previous results [Galizia et al., 2010]. VL2 is almost out of focus and hardly visible in figure 3.9 A, but elicited a clear response towards 1-Oct. As the receptor type of VL2 innervating neurons is not known, a comparison with previous results was not possible. However, the responsiveness towards alcohols described by Rodrigues [1988] is supported by the signal obtained in response to 1-Oct.

Conclusively, the investigation of the response profiles of the three used odorants 1-octen-3-ol (1-Oct), 3-octanol (3-Oct) and 4-methylcyclohexanol (MCH) confirms a similarity of 1-Oct and 3-Oct also on a physiological level in the antennal lobes. The activation patterns evoked by MCH are more distinct. Hence, the structural similarity of 1-Oct and 3-Oct is not only confirmed by a similar perception manifested in a generalization behavior towards the odorants (section 3.4), but also by a physiolog-ical similarity in neuronal activity in the antennal lobes. However, the physiologphysiolog-ical responses towards the two odorants were not completely identical. As shown in figure 3.9, the VL2 glomerulus responded only to a presentation of 1-Oct and not to 3-Oct. As this confirms the assumption that the two odorants are not perceived the same, it was investigated if the flies could be trained to discriminate between the two similar odorants.

3. Results