A novel Similarity Analysis Demonstrates the Functional Independence of γ-Lobe

only a single odor but showed varying combinations of odor responses (Fig. 3.8).

Figure 3.8: Median response profiles of 31 γ-KCs. Each row shows a section of the time trace (30 frames (7.25 s) 5 s after imaging onset) of the ΔF/F0 [%] values false color coded as indicated in the calibration bar (right). Each panel corresponds to a γ-lobe compartment grouped by odor stimulation. Grey bars underneath indicate odor stimulus window. The three arrow heads on the left refer to the γ-KCs in Fig. 3.6 and Fig. 3.7.

3.4.2 A novel Similarity Analysis Demonstrates the Functional Independence of γ-Lobe Compartments

The data has shown that γ-KCs exhibit γ-lobe compartment-specific profiles, but are these compartments functionally independent units? To address this question a new analysis was introduced (see 2.2.7). This analysis compared the similarities between γ-lobe compartments.

First, the peak responses (average of 5 time frames, see Methods 2.2.6) for each of the 1945 boutons were calculated. To compare the activity between the γ-lobe compartments, the median of each responding compartment was calculated. This resulted in 45 odor responses per γ-lobe compartment. Even though each γ-KC responded individually along γ-lobe compartments, the median responses showed no differences between compartments (Fig. 3.9 a). Only a slight tendency was observed for γ4 and γ5 showing weaker responses. Therefore, the second step was to calculate the normalized cross-correlation coefficient between the boutons of a γ-KC within (internal) a compartment and between (external) compartments (Fig. 3.9 b). Boutons that showed responses to an odor were highly correlated even though their peak intensities varied in magnitude (Fig. 3.9 b). To compare the correlations between γ-lobe compartments, the median

67 correlation coefficient was calculated for each compartment in each γ-KC. Here as well, the compartments that were responding showed high correlations and did not reflect the differences in amplitude (Fig 3.8 e). To combine both features of the bouton responses and compare the similarities of γ-lobe compartments the cross-correlation coefficients of each bouton pair was multiplied by the amplitude contrast of the respective bouton pair (see methods 2.2.7). The bouton responses were subsequently normalized to the maximum of the response, to be able to compare the values between γ-KCs. This amplitude corrected correlation (ACC) index reflected the pattern of similarity within the γ-lobe compartment (Fig. 3.9 f, highlighted diagonal matrix fields) and between compartments (Fig. 3.9 f, remaining matrix fields).

Figure 3.9: Activity corrected correlation as a new measure for similarity. a Box-plot shows the 45 median responses of the 31 γ-KCs. No significant difference (n. s.) was found between the γ-lobe compartments.

There is a tendency for γ4 and γ5 being lower in intensity than γ2 and γ3. b Single bouton calcium dynamics of an exemplary γ-KC (KC1, yellow, in Fig. 3.4) responding to MCH stimulation. Colored time traces correspond to the respective γ-lobe compartment. Different boutons responded in different intensities to the same stimulation. Red dashed line indicates the correlation window that was used for further analysis. c Matrix showing the normalized cross-correlation coefficient (norm. c.c.) of the time traces of each bouton in b. As all boutons showed an increase in intensity, the norm. c.c. were high (red). The norm. c.c. could not reflect the differences in the peak intensities (b). Colored bars left and beneath the matrix indicate which boutons belong to which γ-lobe compartment. d Median calcium dynamics of b for the four γ-lobe compartments. On average all compartments showed a response to the MCH stimulation with different peak intensities. e Median norm. c.c. of c plotted in a matrix showing the γ-lobe compartments’ internal correlation on the diagonal (highlighted in thick black contours) and the correlations between compartments. Here as well, the differences in peak intensities (d) could not be reflected. f After introduction of the amplitude corrected correlation (ACC, see methods 2.2.7) index the matrix could reflect the strength of similarities (color map from black to red).

68

With the ACC index it was now possible to visualize the relationships between the γ-lobe compartments and address the question if they are functionally independent units. First, the medians of all ACC indices were plotted in a colored matrix (Fig. 3.10 a, values in Tab. 3.2). This matrix showed that the naïve odor responses, of the 31 γ-KCs (45 odor responses) measured, were more similar within a compartment than between them. Specifically, γ2 and γ3 showed the highest internal similarities (yellow ACC index), thereby grouping together. The γ-lobe compartments 4 and 5 had lower internal similarities (cyan ACC index), thereby also grouping together. This tendency was comparable to the trend in the median bouton fluorescence intensities (Fig. 3.9 a). In order to determine the functional independence of the γ-lobe compartments, the contrast of the γ-lobe internal ACC index to the external ACC indices was calculated. If the median contrast would have resulted in 0 there would have been no difference between the internal and the external similarities. This would have meant that the boutons of one γ-lobe compartment were not distinguishable from boutons of other compartments. For γ2 – γ4 the 95 % CI of the median ACC index contrasts were above the 0-value. Therefore, it could be demonstrated that the γ2-, γ3-, and γ4-compartment can be assumed to be functionally independent units (Fig. 3.10 b). In contrast, the bouton-similarities in the γ5-compartment were not distinguishable from boutons of other γ-lobe compartments. This finding demonstrates for the first time that γ-KCs are not binarily coding for an odor by simply being active or not active.

They comprise functional units that could be independently active and might code for different modalities of a stimulus. This finding demonstrates that the number of coding combinations is increased by the number of γ-lobe compartments. Hence, it increases the odor coding space enormously.

Figure 3.10: γ-lobe compartments are functionally independent units. a Median similarity (ACC index) of all 31 γ-KCs illustrated as color coded matrix (values in Tab. 3.2). Diagonal indicates the internal similarity (highlighted as black contour). Remaining fields indicate the external similarities between γ-lobe compartments. b Contrast of the internal ACC index to the external ACC index. The circles indicate the median ACC index contrast; whiskers indicate the 95 % CI. The CIs of γ2-γ4 do not cross the 0-value, resulting in a 5 % significance being different from 0.

69 Table 3.2: Median ACC indices of the 31 γ-KCs plotted in Fig. 3.10

γ2 γ3 γ4 γ5

γ2 0.60431 0.5233 0.38328 0.30769 γ3 0.5233 0.60462 0.34005 0.31706 γ4 0.38328 0.34005 0.46602 0.39657 γ5 0.30769 0.31706 0.39657 0.4014