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Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination

Nature Neuroscience volume 17, pages 559–568 (2014)Cite this article

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Abstract

Sparse coding may be a general strategy of neural systems for augmenting memory capacity. In Drosophila melanogaster, sparse odor coding by the Kenyon cells of the mushroom body is thought to generate a large number of precisely addressable locations for the storage of odor-specific memories. However, it remains untested how sparse coding relates to behavioral performance. Here we demonstrate that sparseness is controlled by a negative feedback circuit between Kenyon cells and the GABAergic anterior paired lateral (APL) neuron. Systematic activation and blockade of each leg of this feedback circuit showed that Kenyon cells activated APL and APL inhibited Kenyon cells. Disrupting the Kenyon cell–APL feedback loop decreased the sparseness of Kenyon cell odor responses, increased inter-odor correlations and prevented flies from learning to discriminate similar, but not dissimilar, odors. These results suggest that feedback inhibition suppresses Kenyon cell activity to maintain sparse, decorrelated odor coding and thus the odor specificity of memories.

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Figure 1: Feedback inhibition of Kenyon cell responses by Kenyon cell output.
Figure 2: Feedback is from all Kenyon cells to all Kenyon cells.
Figure 3: Kenyon cells activate APL.
Figure 4: APL inhibits Kenyon cells.
Figure 5: Inhibition keeps Kenyon cell responses sparse and distinct.
Figure 6: APL sparsens and decorrelates Kenyon cell responses.
Figure 7: Feedback inhibition facilitates learned discrimination of similar, but not dissimilar, odors.
Figure 8: Partial effect of APL-specific RNAi of GABA biosynthesis.

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Acknowledgements

We thank M. Parnas and S. DasGupta for discussions; Y. Tan, R. Roorda, C. Talbot and J. Beevers for technical assistance; L. Looger (Janelia Farm Research Campus) for a plasmid encoding GCaMP3; and S. Goodwin (University of Oxford), C.-H. Lee (US National Institutes of Health), L. Luo (Stanford University), K. Scott (University of California, Berkeley), S. Waddell (University of Oxford), the Bloomington Stock Center, the Kyoto Drosophila Genetic Resource Center and the Vienna Drosophila RNAi Center for fly strains. This work was supported by grants from the Wellcome Trust (090309/Z/09/Z; G.M.), the Gatsby Charitable Foundation (G.M.), the UK Medical Research Council (G0700888; G.M.), the US National Institutes of Health (R01DA030601; G.M.), the Oxford Martin School (G.M.), the Howard Hughes Medical Institute (T.L.), a Sir Henry Wellcome Postdoctoral Fellowship (089021/Z/09/Z; A.C.L.) and a Wellcome Trust OXION studentship (A.M.B.).

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  1. Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK

    Andrew C Lin, Alexei M Bygrave, Alix de Calignon & Gero Miesenböck

  2. Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Tzumin Lee

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Contributions

A.C.L. and G.M. designed the study, analyzed the results and wrote the paper. A.C.L. performed all behavioral and imaging experiments. A.M.B. assisted with behavioral experiments and dissections and A.d.C. with dissections. T.L. provided lexAop-shits1 and mb247-LexA flies.

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Integrated supplementary information

Supplementary Figure 1 GAL4 driver lines labeling α'β' and γ Kenyon cells.

(a,b) Maximum intensity z-projections of confocal image stacks. (a) γ Kenyon cells are labeled in R64C08-GAL4/UAS-CD8::GFP flies. (b) α'β' Kenyon cells are labeled in R35B12-GAL4/UAS-CD8::GFP flies. Scale bars, 50 μm. Representative images selected from at least 3 dissected brains.

Supplementary Figure 2 Intersectional strategy allows specific labeling of APL neurons with insertions of tubP-FRT-GAL80-FRT on chromosomes II and III.

(a) Anterior view of GFP expression in NP2631-GAL4, GH146-Flp/tubP-FRT-GAL80-FRT,UAS-CD8::GFP; UAS-shits1/+ fly with both APL neurons labeled. (b) Anterior view of GFP expression in NP2631-GAL4, GH146-Flp/UAS-CD8::GFP; tubP-FRT-GAL80-FRT/+ fly with both APL neurons labeled. Scale bars, 50 μm. Representative images selected from 10 (a) and 5 (b) confocal stacks and nearly 700 brains dissected (see Fig. 6).

Supplementary Figure 3 APL inhibits Kenyon cells in all lobes.

(a-d) Impact of different APL manipulations (rows) on odor-evoked Ca2+ influx in different mushroom body lobes (columns). Black bars indicate 5 s pulses of ethyl acetate. (a) In control hemispheres of APL>shits1 flies where APL was unlabeled, odor-evoked Ca2+ influx in Kenyon cells declined slightly at 32 °C. (b) In hemispheres of APL>dTRPA1 flies where APL was labeled, odor-evoked Ca2+ influx in Kenyon cells was almost completely abolished at 32 °C. (c) In hemispheres of APL>shits1 flies where APL was labeled, odor-evoked Ca2+ influx in Kenyon cells increased greatly at 32 °C. (d) In APL-labeled hemispheres of APL>TeTx flies (red), odor-evoked Ca2+ influx in Kenyon cells was much higher than in APL-labeled hemispheres of APL>TeTx-inactive flies (blue). (e-h) Bar graphs summarizing maximum ΔF/F from (a-d) and including data after recovery to 22 °C after heating (e-g) and hemispheres from APL>TeTx flies where APL was unlabeled (green bars, h). n, left to right, given as number of brain hemispheres [number of flies]: (e) n=24 [17] (α and α'); 7 [6] (β, β', γ) (f) n=9 [5], 8 [5]. (g) n=30 [21] (α and α'); 15 [10] (β, β', γ). (h) n=9 [7], 10 [8], 7 [6] (α and α'); 10 [6], 7 [6] (β, β', γ). (i) Ratios of odor-evoked Ca2+ influx at 32 °C vs. 22 °C, corresponding to panels e-h. Error bars show s.e.m. * P<0.05, ** P<0.01, *** P<0.001, repeated-measures ANOVA with Geisser-Greenhouse correction and Holm-Sidak multiple comparisons test (e,f – α, g – α', β, β', γ), Friedman test with Dunn's multiple comparisons test (f – α', g – α), one-way ANOVA with Tukey-Kramer post hoc test (h – α, α'), unpaired Welch t-test (h,i – β, β', γ) or Mann-Whitney U test (i – α, α'). See Supplementary Table 1 for full genotypes.

Supplementary Figure 4 Kenyon cell expression of TeTx is required for the effect of OK107>TeTx on sparse coding.

(a) Activity maps of odor responses in OK107>TeTx, mb247-LexA>GCaMP3,GAL80 flies (left, n = 7 [5]) and OK107>TeTx, mb247-LexA>GCaMP3 (right, n = 6 [6]). Color-coded correlation matrices as in Fig. 5a,b. Scale bars, 10 μm. n given as number of hemispheres [number of flies]. (b, c) Removing Kenyon cell expression with mb247-LexA>GAL80 increases the population sparseness (b) and decreases the inter-odor correlations (c) of activity maps of odor responses in flies carrying OK107>TeTx. * P < 0.05, unpaired Welch t-test. See Supplementary Table 1 for full genotypes.

Supplementary Figure 5 Blocking APL synaptic output affects Kenyon cell responses to broad odors more than Kenyon cell responses to narrow odors.

ΔF/F traces and maximum ΔF/F data corresponding to the ratios in Fig. 6a. Shading on traces indicates s.e.m. Black bars indicate 5-s pulses of δ-DL, IA:EB 1:4, or IA:EB 4:1 as labeled. * P < 0.05, ** P < 0.01, *** P < 0.001, repeated-measures ANOVA with Geisser-Greenhouse correction and Holm-Sidak multiple comparisons test. n = 12 [8] (APL>shits1 labeled), n = 6-7 [5] (APL>shits1unlabeled). n given as number of hemispheres [number of flies].

Supplementary Figure 6 Learned odor discrimination of flies with only one APL neuron labeled and with OCT vs. MCH.

(a) Learned odor discrimination of 3-octanol (OCT) vs. 4-methylcyclohexanol (MCH). Protocol was as in Fig. 7a except the CS+ was OCT and the CS– was MCH. n, left to right, given as number of flies [number of experiments]: 29 [6], 14 [6], 19 [6], 41 [6], 25 [6], 18 [6], 16 [6], 32 [6]. Kruskal-Wallis ANOVA reveals no significant differences between the 8 groups (P = 0.09). 2-way ANOVA reveals a main effect of temperature (P < 0.05) but no significant interaction between genotype and temperature across flies with neither, left, right, or both APL neurons labeled (P = 0.52) or comparing flies with neither or both APL neurons labeled (P = 0.82). (b) Data as in Fig. 7c, adding single-APL-labeled flies. CS+ was IA:EB 4:1 and CS– was δ-DL (‘dissimilar’) or IA:EB 1:4 (‘similar’). At 32 °C, flies with only the left or right APL labeled by shits1 are somewhat impaired on learned discrimination of similar odors compared to flies with neither APL labeled, but the difference is not statistically significant. n, left to right, given as number of flies [number of experiments]: 23 [6], 28 [6], 19 [6], 26 [6], 16 [7], 32 [7], 34 [7], 44 [7], 18 [8], 30 [8], 22 [8], 55 [8], 32 [9], 33 [9], 37 [9], 51 [9]. * P < 0.05, ** P < 0.01, *** P < 0.001, Kruskal-Wallis ANOVA with Holm-Bonferroni correction for post hoc tests, testing only pairs of data points with one variable changed (task, temperature, and APL labeling). P < 0.05, 3-way ANOVA for interaction of task, temperature, and APL labeling. P < 0.01, 2-way ANOVA for interaction of genotype and temperature for discrimination of similar odors. P < 0.05, 2-way ANOVA for interaction of task and APL labeling at 32 °C. P < 0.05, 2-way ANOVA for interaction of task and temperature for flies with both APL neurons or the left APL neuron labeled. Other 2-way ANOVAs did not reveal any significant interactions. Error bars show s.e.m.

Supplementary Figure 7 RNAi knockdown of GABA biosynthesis in the APL neuron causes only partial effects on Kenyon cell odor responses.

(a–d) See grid at bottom for full genotypes. (a) α' lobe responses to ethyl acetate. (b) a lobe responses to ethyl acetate. (a,b) n, left to right, given as number of brain hemispheres [number of flies]: 25 [17], 30 [21], 9 [7], 10 [8], 21 [19], 22 [19], 10, 11, 6, 6. n given as number of hemispheres [number of flies]. (c) Mean population sparseness of cell body responses to the panel of odors used in Fig. 5. (d) Mean inter-odor correlations between cell body responses for the panel of odors used in Fig. 5. (c,d) n, left to right, given as number of brain hemispheres [number of flies]: 7 [7], 8 [7], 9 [7], 9 [7], 8 [8], 10 [8], 10, 11, 5, 5. (e) Sample activity maps of cell body responses analyzed in panels c and d. Compare to Fig. 5a,b. Scale bars, 10 μm. Color grids represent pairwise correlations as in Fig. 5: 1) ethyl acetate, 2) 3-octanol, 3) butyl acetate, 4) isoamyl acetate, 5) ethyl butyrate, 6) 2-pentanol, 7) 4-methylcyclohexanol. * P < 0.05, ** P < 0.01, *** P < 0.001 significant difference between colored bars and the relevant controls (gray bars), by unpaired Welch t-test for APL>shits1 and APL>GADRNAi and by Kruskal-Wallis ANOVA and Dunn’s multiple comparisons test for GH146 and NP2631 driving GADRNAi. § P < 0.05 significant difference between effect of GADRNAi and effect of APL>shits1 by 2-way ANOVA. † P < 0.05 significant difference between effect of manipulation marked with † and effect of APL>TeTx by 2-way ANOVA. # P < 0.05 significant difference between effect of manipulation marked with # and effect of APL>GADRNAi by 2-way ANOVA. Error bars show s.e.m.

Supplementary Figure 8 RNAi knockdown of GABA biosynthesis in the APL neuron does not affect sparseness or correlation of Kenyon cell responses to MCH or OCT.

(a-c) See grid at bottom for full genotypes. (a) Population sparseness of cell body responses to 4-methylcyclohexanol (MCH). Statistical comparisons, left to right: Mann-Whitney U test (P = 0.094), unpaired Welch t-test (P = 0.50), one-way ANOVA (P = 0.48). (b) Population sparseness of cell body responses to 3-octanol (OCT). Statistical comparisons: Mann-Whitney U test (P = 0.054), unpaired Welch t-test (P = 0.49), Kruskal-Wallis ANOVA (P = 0.41). (c) Correlation between cell body responses to MCH and OCT. Statistical comparisons: unpaired Welch t-test (P = 0.10), Mann-Whitney U test (P = 0.75), Kruskal-Wallis ANOVA (P = 0.34). n, left to right, given as number of brain hemispheres [number of flies]: 7 [7], 8 [7], 8 [8], 10 [8], 10, 11, 5, 5. Error bars show s.e.m.

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Lin, A., Bygrave, A., de Calignon, A. et al. Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination. Nat Neurosci 17, 559–568 (2014). https://doi.org/10.1038/nn.3660

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