Timing of CS- presentation has no obvious influence on learning
3.11. Changes in neuronal activity in the mushroom body as a result to learning: first steps
Neuronal activity in the mushroom body was assessed by measuring the changes in calcium concentrations in response to odorant presentation with GCaMP3.0 [Tian et al., 2009]. As theβ’ and theγ-lobe were implicated in memory acquisition [Krashes et al., 2007; Qin et al., 2012] and dopaminergic neurons responsible for aversive conditioning innervate theγ-lobe [Aso et al., 2010; Qin et al., 2012; Waddell, 2013], a focal plane that includes theβ’- and theγ-lobe was selected. As an additional land-mark, a protuberance in the γ-lobe could be identified (for a reconstruction of the mushroom body and the focal plane selected for calcium imaging, see figure 3.24 A and B). An identification of the two lobes included in the experiment was possible due to a higher basal fluorescence observed in theγ-lobe. Changes in calcium con-centrations as a correlate for neuronal activity was measured in response to three pseudo-randomized presentations of the two similar odorants 1-Oct and 3-Oct and the control odorant MCH (for a scheme of the whole imaging procedure, see 2.4).
Signals could be observed for all three odorants in the β’-lobe and in the medial regions of the γ-lobes, referred to by Tanaka et al. [2008] as γ1, γ2 and γ3 (false-color coded images of an example fly are depicted in figure 3.24 C). The average odorant responses of the whole mushroom body for the example fly during odorant presentation are depicted in figure 3.24 D. The elicited responses were consistent for each of the three odorant presentations. In order to compare the spatial
activ-3. Results
Figure 3.24. Neuronal activity pattern in the mushroom body.
(A) A 3D-reconstruction of the mushroom body shows the different lobes (see sec-tion 1.4.3 and figure 1.5). d = dorsal; a = anterior; m = medial(B)The focal plane for the imaging experiments is depicted in gray. Calcium activity could therefore be measured from theγand theβ’-lobes. The two lobe regions were differentiated by the difference in the basal fluorescence as shown on the right side (theγ-lobe consistently showed a higher basal fluorescence than β’). (C) False-color coded images of the evoked calcium responses during odorant presentation.(D)Average responses from three calcium measurements with the same odorants in one fly in the whole mush-room body.(E)Pixel based correlation coefficient calculated from the average odorant responses for the three possible combinations in one fly.(F)The correlation coefficient between the two similar odorants 1-Oct and 3-Oct from 20 measured flies showed a significantly higher correlation compared to the correlation between 1-Oct and MCH
(and 3-Oct and MCH, respectively). Scale bar = 20µm
Friedman ANOVA with post hoc Bonferroni corrected paired sample Wilcoxon signed rank test; *** = p < 0.001
ity patterns evoked by the odorants, the correlation between the patterns for each possible odorant pair was calculated from the average responses for each fly (the correlation coefficients of the example fly are shown in figure 3.24 E). Comparable with the responses evoked in the antennal lobe (see section 3.5), a correlation be-tween 1-Oct and 3-Oct could also be observed in the mushroom body. The neuronal activity patterns elicited by 1-Oct and 3-Oct from 20 measured flies before any con-ditioning showed a stronger correlation than the pattern evoked by 1-Oct and MCH and 3-Oct and MCH, respectively (Figure 3.24 F). It can therefore be stated that the similarity of the two odorants that was observed on the behavioral level and on the physiological level in the antennal lobe is conveyed further to the mushroom body.
For the subsequent analysis of possible conditioning dependent changes, the mush-room body was subdivided in two regions. As described above, odorant evoked sig-nal could be observed in the β’-lobe and in theγ1,γ2 andγ3 -regions of theγ-lobe which will be referred to as the γ-shaft from now on (Figure 3.25 A). Both regions elicited a signal during odorant presentation in all measured flies (n = 20) as depicted in figure 3.25 B and C. It has to be mentioned that the amplitude of the signal varied between flies. Especially the activity in the γ-lobe was very variable. The 20 flies that were imaged during this experiment were further subdivided into two groups.
One group received an absolute training with either 1-Oct or 3-Oct (5 flies each) as a CS+ and mineral oil was presented instead of a CS-. The second group was trained differentially either with 1-Oct as the CS+ and 3-Oct as the CS- (5 flies) or the other way around (5 flies). The training procedure was the same as during the behavioral experiments with a temporal pairing of the CS+ with 12 electric shocks within one minute and the CS- presented for one minute without reinforcement (see schemes in figure 3.25 D and E). Different effects on the correlation coefficients (as measure for odorant similarity [Svedlow et al., 1976]) could be observed depending on the training paradigm applied. Absolute training on the one hand resulted in a significantly higher correlation of 1-Oct and 3-Oct in the β’-lobe whereas no effect could be observed in theγ-shaft region (Figure 3.25 D). In contrast, differential train-ing did not change the correlation of the two similar odorants in the β’-lobe but a significant decrease in correlation in theγ-shaft region (Figure 3.25 E). Note that the variation of the correlations calculated for the distinct lobes was different. The vari-ability of the correlations in theβ’-lobe was rather low but for the γ-shaft they were
3. Results
Figure 3.25. Learning evoked changes in odorant representation in the mush-room body.
The mushroom body was subdivided into two regions for the analysis.(A)One region consisted of theβ’-lobe and the other the shaft of theγ-lobe. These two regions were chosen by the odorant evoked calcium signals observed during stimulation depicted in figure 3.24 C.(B,C)Averaged time courses of evoked calcium signals before condi-tioning from 20 flies.(D)Comparison of maximal pixel based correlation coefficients of activation patterns evoked by 1-Oct and 3-Oct in the two regions in 10 flies after abso-lute training. A significant increase of the correlation between the two similar odorants can be observed in theβ’-lobe whereas theγ-shaft region did not show a significant effect of absolute training. (E)Comparison of maximal pixel based correlation coeffi-cients of activation patterns evoked by 1-Oct and 3-Oct in the two regions in 10 flies after differential training. No change in theβ’-lobe could be detected. In theγ-shaft, a significant decrease of correlation reflecting a stronger dissimilarity after the training procedure is prominent. Red bars in (D) and (E) represent the average correlation co-efficient. n = 10 ; paired sample Wilcoxon signed rank test
varying stronger. An explanation for this difference can be attributed to the differ-ence in amplitude of the elicited signals. Indeed, the signals obtained in theβ’-lobe were consistently higher than in theγ-shaft region. A further analysis of the outliers observed in figures 3.25 D and E revealed that the outliers are produced by flies with very low signals in the respective regions. An exclusion of the outliers from the statistical analysis did not change the overall outcome of the analysis.
In conclusion, the physiological similarity of 1-Oct and 3-Oct that could be observed with Ca2+-imaging in the antennal lobe is conveyed to the mushroom room body lobes and results in a higher correlation of the elicited neuronal responses. This cor-relation between the activation pattern elicited by 1-Oct and 3-Oct could be further increased in theβ’-lobe after absolute training. Conversely, differential conditioning resulted in a decreased correlation of the evoked pattern in theγ-shaft region of the mushroom body.