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Plasticity-driven individualization of olfactory coding in mushroom body output neurons

Abstract

Although all sensory circuits ascend to higher brain areas where stimuli are represented in sparse, stimulus-specific activity patterns, relatively little is known about sensory coding on the descending side of neural circuits, as a network converges. In insects, mushroom bodies have been an important model system for studying sparse coding in the olfactory system1,2,3, where this format is important for accurate memory formation4,5,6. In Drosophila, it has recently been shown that the 2,000 Kenyon cells of the mushroom body converge onto a population of only 34 mushroom body output neurons (MBONs), which fall into 21 anatomically distinct cell types7,8. Here we provide the first, to our knowledge, comprehensive view of olfactory representations at the fourth layer of the circuit, where we find a clear transition in the principles of sensory coding. We show that MBON tuning curves are highly correlated with one another. This is in sharp contrast to the process of progressive decorrelation of tuning in the earlier layers of the circuit2,9. Instead, at the population level, odour representations are reformatted so that positive and negative correlations arise between representations of different odours. At the single-cell level, we show that uniquely identifiable MBONs display profoundly different tuning across different animals, but that tuning of the same neuron across the two hemispheres of an individual fly was nearly identical. Thus, individualized coordination of tuning arises at this level of the olfactory circuit. Furthermore, we find that this individualization is an active process that requires a learning-related gene, rutabaga. Ultimately, neural circuits have to flexibly map highly stimulus-specific information in sparse layers onto a limited number of different motor outputs. The reformatting of sensory representations we observe here may mark the beginning of this sensory-motor transition in the olfactory system.

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Figure 1: Summary of olfactory tuning patterns in MBONs.
Figure 2: Transformation of population representations from KCs to MBONs.
Figure 3: Individualization of tuning in MBONs.
Figure 4: Connection probability between KCs and a MBON.
Figure 5: Cross-individual variability is lost in rutabaga mutants.

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Acknowledgements

We would like to thank V. Jayaraman, J. Dubnau and K. Ito for fly strains. We are grateful to H. Kazama, W. Li and J. Dubnau for helpful advice, and to V. Jayaraman, G. Otazu and the members of the Turner laboratory for valuable comments on the manuscript. This work was supported by NIH grant R01 DC010403-01A1 to G.C.T.; T.H. was partially supported by a Postdoctoral Fellowship for Research Abroad from Japan Society for the Promotion of Science and a Postdoctoral Fellowship from the Uehara Memorial Foundation.

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Authors

Contributions

T.H. and G.C.T. designed the experiments with help from Y.A. and G.M.R.; T.H. performed all imaging and electrophysiology experiments and data analyses. Y.A. and G.M.R created fly strains and collected anatomical data for MBONs. T.H. and G.C.T. wrote the manuscript.

Corresponding author

Correspondence to Glenn C. Turner.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Comparison of odour-evoked calcium responses across multiple cell compartments.

a, Odour responses were sequentially recorded at three different compartments (axon, blue; dendrite, orange and soma, black) in two different α2sc neurons with GCaMP5 imaging. Upper subpanel shows mean ΔF/F traces from different compartments (4 or 5 trials for each). Shaded areas indicate 1-s odour stimulations. Lower subpanel shows tuning profiles in axon and dendrites (normalized to the strongest response, mean ± s.e.m.). Note that the time course, response magnitude and tuning profiles are largely consistent between the axon and dendrite in the same cell, while signals at the soma are small and slower (Cell2) or sometimes undetectable (Cell1). be, Data from 4 other types of MBONs. Scale of the traces is the same as in a. Correlation between axons and dendrites is 0.92 ± 0.013 (Pearson’s r, mean ± s.e.m.; n = 10 cells). Hya, hexyl acetate. f, Mean normalized tuning of the ten cells. Odours are sorted according to the rank order in each cell to visualize the tuning width. g, Kurtosis of tuning curves. There was no difference between axons and dendrites (P = 0.16, paired t-test).

Extended Data Figure 2 Raw traces of calcium imaging in MBONs.

For all 17 types/combination of types of MBONs, we show a projection image of confocal stacks from split-GAL4 lines (left), example ΔF/F images of the calcium responses to ten odours and air (middle) and ΔF/F time courses (right). Note that all the split-GAL4 lines used in this study label the target MBONs with extremely high specificity. The white squares indicate the approximate region scanned during calcium imaging. Greyscale images show baseline fluorescence (scale bar, 5 μm), while colour map images represent calcium responses (ΔF/F, colour scale bottom right). Traces showing ΔF/F time courses on individual trials (grey; n = 4–7) are overlaid with the mean (red), scale bar, 50% ΔF/F.

Extended Data Figure 3 Relationship between response intensity and tuning selectivity.

Lifetime sparseness, an index of tuning selectivity1,44, was plotted against the magnitude of the largest odour response observed for each neuron (mean ΔF/F during the response window). Each dot corresponds to one of the 17 MBON types (mean ± s.e.m.; n = 5 flies). There was no correlation between the two variables (Spearman’s ρ = −0.16, P = 0.54). Thus, the relatively selective tuning patterns observed in four MBONs (blue) are likely not related to the intensity of the response. Rather, for three of these cells, we noticed that their narrow tuning was accompanied by unusual dendritic anatomy. β′1 neuron is the only MBON with extensive dendritic processes outside the MB, and calyx neuron (MB-CP1) is the only MBON that samples from the MB calyx. α2p3p neuron’s dendrites seem to contact solely the α/βp KCs, whose dendrites arborize in the sub-region of the MB calyx known as the accessory calyx, where no olfactory input has been reported7. The narrow tuning patterns of the fourth neuron, γ5β′2a, seem to be related to the transient inhibitory epochs uniquely observed in this neuron (Extended Data Fig. 4).

Extended Data Figure 4 Diversity in response time courses across the MBON population.

Normalized mean ΔF/F responses to yeast odour for all MBON types overlaid (shading indicates timing of odour presentation). Note that three MBONs (β1, γ1pedc, and γ4) that have axonal terminals in the MB lobes show slower time courses (red) than the others (black). The cell with the characteristic inhibitory period is the γ5β′2a neuron (blue).

Extended Data Figure 5 Population analysis of subpopulations of MBONs.

a, Distribution of MBON terminals in the brain (dark colours), based on the localization of a presynaptic marker8 (Syt::smGFP-HA). Anterior (top) and dorsal (bottom) views are shown. Axonal projections of MBONs converge heavily onto small areas inside four neighbouring neuropils (light colours), crepine (CRE; orange), superior medial protocerebrum (SMP; green), superior intermediate protocerebrum (SIP; purple) and superior lateral protocerebrum (SLP; blue). We have found numerous cells that send widely branching neurites into one or all of these areas (database associated with ref. 45), indicating that downstream neurons likely read out activity from populations of MBONs. Lateral horn (LH, red) also receives sparse input. b, Pairwise correlations of MBON tuning patterns and the corresponding dendrogram are shown in the same way as Fig. 1d. The dots on the right show the axonal projection site of the different MBON types. None of the four major projection zones samples from particularly decorrelated set of MBONs. c, Odour representations in MBONs from a single virtual fly visualized by PCA (different virtual fly from Fig. 2c). Representations from the full MBON population (All) and subpopulations with the same axonal projection zones (CRE, SMP, SIP and SLP) are shown. Representations by subpopulations tend to be noisier than those from the full population, but they retain grossly similar structures. d, Correlation coefficients of neural representations of ten odours in KCs and MBONs (thick lines), as in Fig. 2g, h. Correlation values observed after artificially decorrelating KC and MBON tuning curves are shown for comparison (thin lines). e, Variance of the correlation coefficients shown in d (dark bars; mean ± s.d.; n = 1,000 virtual flies), showing that values are more widely distributed in MBONs compared to KCs. Artificially decorrelating MBON tuning curves reduces the variance considerably (light bars), indicating that the pattern of activity in the MBONs contributes to the high variance. However, variance remains greater than that in the KCs, indicating that the breadth of tuning in the MBONs also contributes to wide range of correlation coefficients. The ratio of the contribution of those two factors seems to be different across subpopulations projecting different zones.

Extended Data Figure 6 Correlation of tuning between multiple MBON types measured in the same animal.

a, Odour tuning of α1 and α2sc neurons recorded sequentially in the same fly with GCaMP5 imaging (normalized to the strongest response, mean ± s.e.m.). Data from five representative flies are shown. Two split-GAL4 drivers, MB319C and MB080C, were combined to label the two cell types. b, Similar to a but recordings are from α1, β1 and γ4 neurons. MB319C was combined with MB434B that labels both β1 and γ4 neurons. c, Correlation of tuning between different cell types, calculated within and across flies. Four different pairwise combinations of cell types were examined. Although within-fly correlations tended to be slightly higher than across-fly correlations, none of the four cases was significant (Mann–Whitney U-test). This is in sharp contrast with the comparisons of the same cell type, where we observed much higher correlations within the same fly than across flies (Fig. 3).

Extended Data Figure 7 Confusion matrices from odour classification analysis.

Confusion matrices generated from the classification analysis in Fig. 2d. a, KCs show nearly perfect classification performance. b, MBONs fail to discriminate some odours. Although the confusion matrix was generated using 100 different virtual flies (see Methods), a high rate of misclassifications is observed only between certain odour pairs that form overlapping clouds in MBON space (Fig. 2c). Odour pairs that were never misclassified with each other are indicated with white asterisks. Note that the group of vinegar and yeast were never misclassified with the group of CO2 and citronella.

Extended Data Figure 8 Individualized tuning in multiple different MBON types.

ac, Odour tuning of pairs of MBONs in the same fly, from GCaMP5 imaging at axons (normalized to the strongest response, mean ± s.e.m.). Data from five representative flies are shown. For γ1pedc (a) and α′2 neurons (b), cells on the left and right hemispheres are compared. For α1 neuron (c), recordings were from ipsilateral cell pairs. df, For all three cell types, tuning patterns of the neurons from the same fly are more correlated than those from different flies (n = 8 flies per cell types; Mann–Whitney U-test).

Extended Data Figure 9 Diverse functional connections between KCs and a MBON.

a, Another example of a monosynaptically connected pair of α/β KC and the α2sc neuron from experiments shown in Fig. 4. a1, Sample traces of simultaneously recorded KC (Pre) and the α2sc neuron (Post). a2, The α2sc neuron’s membrane potential spike-trigger-averaged on the first KC spike of each trial. Raster plot of the KC spikes is shown below. a3, Enlarged spike- trigger-averaged EPSPs for the first, second and third spikes in the train. b, A pair with small excitatory connection that we could not confirm as monosynaptic. In total, two of such pairs were found. c, A pair with an inhibitory connection, which is likely to be polysynaptic. Only one such pair was found. d, Summary of all five monosynaptically connected pairs. The spike-trigger-averaged EPSPs from the first spike in the train from five different recordings are shown overlaid in grey. The mean across cells is shown in red.

Extended Data Table 1 MBON types and imaging conditions.

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Hige, T., Aso, Y., Rubin, G. et al. Plasticity-driven individualization of olfactory coding in mushroom body output neurons. Nature 526, 258–262 (2015). https://doi.org/10.1038/nature15396

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