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The complete connectome of a learning and memory centre in an insect brain

Abstract

Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higher-order circuit supporting associative memory has not been previously available. Here we reconstruct one such circuit at synaptic resolution, the Drosophila larval mushroom body. We find that most Kenyon cells integrate random combinations of inputs but that a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections found between output neurons could enhance the selection of learned behaviours. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory centre.

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Figure 1: Mushroom bodies of a first-instar Drosophila larva.
Figure 2: KC connectivity reduces redundancy and optimizes stimulus discrimination.
Figure 3: A canonical circuit in every MB compartment.
Figure 4: MBON inputs and circuits.
Figure 5: Intra- and inter-compartment feedback: MBONs of one MB compartment synapse onto MBINs of the same or other compartments.

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Acknowledgements

We thank A. Khandelwal, J. Lovick, J. Valdes-Aleman, I. Larderet, V. Hartenstein, A. Fushiki, B. Afonso, P. Schlegel and M. Berck for reconstructing 31% of arbor cable and 19% of synapses. We thank R. Axel, G. M. Rubin and Y. Aso for their comments on the manuscript. A.L.-K. was supported by National Institutes of Health grant number F32DC014387. A.L.-K. and L.F.A. were supported by the Simons Collaboration on the Global Brain. L.F.A. was also supported by the Gatsby, Mathers, and Kavli Foundations. C.E.P. and Y.P. were supported by the Defense Advanced Research Projects Agency XDATA program (Air Force Research Laboratory contract FA8750-12-2-0303) and National Science Foundation BRAIN EAGER award DBI-1451081. K.E. and A.S.T. thank the Deutsche Forschungsgemeinschaft, TH1584/1-1, TH1584/3-1; the Swiss National Science Foundation, 31003A_132812/1; the Baden Württemberg Stiftung; Zukunftskolleg of the University of Konstanz; and Deutscher Akademischer Austauschdienst. B.G. and T.S. thank the Deutsche Forschungsgemeinschaft, CRC 779, GE 1091/4-1; the European Commission, FP7-ICT MINIMAL. We thank the Fly EM Project Team at Howard Hughes Medical Institute (HHMI) Janelia for the gift of the electron microscopy volume, the Janelia Visiting Scientist program, the HHMI visa office, and HHMI Janelia for funding.

Author information

Authors and Affiliations

Authors

Contributions

K.E., F.L., A.L.-K., B.G., L.F.A., A.S.T., M.Z. and A.C. conceived the project, analysed the data, and wrote the manuscript. K.E., F.L., I.A., C.S.-M., T.S., A.S.T. and A.C. reconstructed neurons. K.E. performed learning experiments. A.L.-K. built the models. J.W.T. contributed GAL4 lines and their imagery. R.D.F. generated electron microscopy image data. A.L.-K., C.S.-M., Y.P. and C.E.P. analysed connectivity patterns. F.L. and A.H. performed immunostainings. C.E. generated functional data.

Corresponding authors

Correspondence to L. F. Abbott, Andreas S. Thum, Marta Zlatic or Albert Cardona.

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Extended data figures and tables

Extended Data Figure 1 Connectivity of the larval APL neuron.

a, Morphology of the right-hemisphere larval APL neuron. While APL dendrites are postsynaptic to the KCs in LA, LVL, IVL, and all medial lobe compartments, its axon is both post- and presynaptic to the MB calyx. Presynaptic sites in red and postsynaptic sites in blue. b, APL connectivity with KC types. Connections are displayed as fractions of input onto the receiving neurons. APL forms more axo-dendritic connections with multi-claw than with single-claw KCs. All mature KCs connect to APL on its dendrite as well as on its axon. c, Strength of synaptic connections between KCs and the APL neuron for single-claw and multi-claw KCs separately. While single-claw KCs have a higher synapse count connecting to the APL dendrites than multi-claw KCs, both groups of KCs project a similar number of synapses to the APL axon. In the calyx, APL makes more synapses onto multi-claw than onto single-claw KCs. In the lobes, single-claw KCs make more synapses with APL pre- than postsynaptically.

Extended Data Figure 2 Input onto KC dendrites and KC–KC connections.

a, Total synaptic input onto KC dendrites from PNs, the APL neuron, and calyx MBINs for single- and multi-claw KCs from both hemispheres. Insert: sum of the KC postsynaptic sites in the calyx as a function of the number of claws. Grey circles show individual KCs and black line shows the mean. Young KCs that have no claws or only form short branches in the calyx have few postsynaptic sites. Mature KCs forming one to six claws present a similar total amount of postsynaptic sites in the calyx. b, A PN bouton (blue) and its associated KC dendritic arborizations. Stars indicate all dendrites of the single-claw KC for this PN. c, Prototypical examples of KCs according to the structure of their dendrites, commonly known as ‘claws’. In first instar, we define each ‘claw’ (indicated by arrowheads) as a connection from a PN providing at least 10% of the postsynaptic sites of the KC dendritic arbor. This connectivity-based definition generally agrees with the count of physically separate claw-like dendritic branches that wrap around the axon terminals of the PNs. Single-claw KCs have not yet been described in the adult fly or other insects. dg, Frequency of different numbers of postsynaptic KC-partners of KCs with at least two-synapse connections, plotted separately for three different types of KC. d, Postsynaptic partners of single-claw KCs. e, Postsynaptic partners of multi-claw KCs. f, Postsynaptic partners of young KCs. g, KC input onto KCs as a percentage of total KC input. For most KCs, more than 50% of their presynaptic partners are other KCs. h, Morphology of an example reconstructed KC (left) and example electron micrographs showing KC–KC synapses (right). Dendro-dendritic connections are in the calyx compartment (1 and 2) and axo-axonic connections are located in the peduncle (3), vertical lobe (4), and medial lobe (5).

Extended Data Figure 3 Test of structure in PN-to-KC connectivity.

a, We performed principal component analysis on the PN-to-KC connectivity matrices for the left and right hemispheres. The analysis was restricted to multi-claw KCs. The variance explained by each principal component in descending order is compared with that obtained from random models in which KCs sample PNs according to the individual PN connection probabilities (see Methods). Grey circles and bars denote mean and 95% confidence intervals for the variance explained by each principal component of the random connectivity matrices, while the black circles indicate the values obtained from data. Note a small deviation from the random model, probably because of the non-olfactory PNs. b, Same as a but restricted to connections between olfactory PNs and KCs. c, Same as b but combining the PN-to-KC connectivity for both hemispheres. The data are compared with a random model in which KCs in each hemisphere sample PNs randomly and independently (grey), and with a bilaterally symmetrical model in which the connectivity is random but duplicated across the two hemispheres (pink). d, To assess the ability of our method to identify structured connectivity, we also generated connectivity matrices in which a weak bias was added (blue). Networks were generated in which PNs were randomly assigned to one of two groups. For each KC, the probabilities of connecting to PNs belonging to one randomly chosen group were increased by 1%, while the probabilities for the other group were decreased by 1% (the baseline probabilities were on average approximately 5%). This procedure was performed independently for each KC and led to networks in which KCs preferentially sampled certain PNs. The data are inconsistent with this model, illustrating that biases of 1% connection probability can be identified using our methods. e, As an independent method to identify structure in the PN-to-KC wiring, we considered the distribution of olfactory PN overlaps for all KC pairs, defined as the number of olfactory PNs from which both KCs received input. This quantity specifically identified biases in the likelihood of KCs to sample similar inputs. As in b, no such structure was identified.

Extended Data Figure 4 KC–KC clustering.

Synaptic connectivity between KCs reveals two communities within the MB. a, Heat map representation of the KC–KC network adjacency matrix, sorted by community structure as discovered by the Louvain method80, which identifies groups of KCs with more within-group connections than expected by chance. We denote the denser community on each side as ‘Group 1’ and the other community as ‘Group 2’. Number of cells in each group is shown in the column labels. b, The number of observed 2+ synapse connections between pairs of KCs within and between groups in the same side of the body, normalized by the total number of all possible such connections (L, left; R, right). c, Distribution of number of synapses per edge for connections within and between each group. Boxes indicate interquartile interval, whiskers the 95th percentile, cross indicates median, outliers shown. d, Claw distribution by group (both sides aggregated). Note that all single-claw KCs are in Group 1. e, Total number of anatomical input synapses onto Group 1 and Group 2 KCs, including from non-KC sources. Group 1 cells have significantly more inputs than Group 2 cells on each side (P < 10−10, t-test with Bonferroni correction). f, Total number of anatomical output synapses from Group 1 and Group 2 KCs, including synapses onto non-KC targets. Group 1 cells have significantly more outputs than Group 2 cells on each side (P < 10−10, t-test with Bonferroni correction). g, KC anatomy labelled by group for the right MB. Note that both groups of KCs come from each lineage cluster. Labelled spots indicate locations of cross-section views in h. h, Cross-sections of the two principal axon branches of KCs in the left (above) and right (below) MBs at the locations indicated in g. Orientations of each cross-section are arbitrary.

Extended Data Figure 5 Neurons in the canonical circuit in every MB compartment.

Neuronal morphology and connectivity of MBINs and MBONs participating in the canonical circuit motif of each MB compartment (MBINs in green and MBONs in magenta). Neurons connecting to the left-hemisphere MB are displayed in anterior view (right hemisphere has the same morphology, data not shown). Locations of MBINs synapsing on MBONs in a given compartment are shown depending on their location (inside the MB neuropil, black; outside the MB, orange). MBIN axons and MBON dendrites tile the MB into 11 distinct compartments. For each compartment, the MBIN and MBON neurons and their connections as a fraction of total input to the receiving neuron are shown on the right. MBONs are diverse in the neurotransmitter that they release (see legend). All compartments present the canonical circuit motif except for SHA, which does not develop its DAN until later in larval life.

Extended Data Figure 6 Fractions of postsynaptic inputs by cell type.

a, Fractions of synaptic output from MBINs onto KCs, other MBINs, MBONs, and other neurons. Some MBINs show a high percentage of connections to MB neurons while others connect with less than 50% of their synapses to MB neurons. b, Number of MBIN–KC synapses in relation to the number of total synaptic outputs from MBIN axons, showing a positive correlation between presynaptic sites on the MBIN axon and synapses dedicated to KC population. c, Number of KC–MBON synapses in relation to the number of total synaptic inputs to MBON dendrites, showing a positive correlation between postsynaptic sites on the MBON dendrite and fraction of input from KCs.

Extended Data Figure 7 MBIN presynaptic vesicle types.

Examples of electron micrographs of MBIN presynaptic sites and vesicles. We found three types of vesicle: large dense-core, small dense-core, and small clear vesicles. Octopaminergic and dopaminergic neurons contain small clear vesicles in addition to other vesicle types. While OANs have all the same type of large dense-core vesicles, DANs show a variety of small dense-core vesicles. We found small dense-core vesicles in one-third of KCs. Some of these were single-claw, others were multi-claw, some received olfactory input, and others non-olfactory PN input. The largest number of dense-core vesicles was observed in the two thermosensory KCs. Scale bar, 500 nm in all panels.

Extended Data Figure 8 Dense-core vesicles in OANs and DANs.

a, Morphology of OAN-a1 and -a2 innervating the calyx of both MBs. b, Location of presynaptic sites (red) and dense-core vesicles (DCVs/black) along the axon of OAN-a1 and -a2. c, DCVs colour-coded by their distance to the closest presynaptic site on the axon of OAN-a1 and -a2. d, Distance (in micrometres) of DCVs to the closest presynaptic site sorted by the value for OAN-a1 and -a2. Most DCVs are within 2 μm from a presynaptic site, just a few are further away and appear to be in transit. e, Presynaptic sites colour-coded by their distance to the closest DCV on the axon of OAN-a1 and -a2. f, Distance (in micrometres) of presynaptic sites to the closest DCV sorted by the value for OAN-a1 and -a2. Half of the presynaptic sites have a DCV associated within 20 μm. Some presynaptic sites have no close DCV associated and are located in the dendrites of OAN-a1 and -a2 (data not shown). g, Morphology of DAN-i1 right innervating the upper toe of the MB medial lobe in both hemispheres with the location of presynaptic sites (red) and DCVs (black). h, Zoom-in onto the medial lobes from g. i, DCV colour-coded by their distance to the closest presynaptic site on the axon of DAN-i1 right. j, Presynaptic sites colour-coded by their distance to the closest DCV on the axon of DAN-i1 right. k, Distance (in micrometres) of DCVs to the closest presynaptic site sorted by the value for DAN-i1 right and left together. Some DCVs are further away from presynaptic sites than 10 μm; these DCVs are in the dendrites of the DAN (shown in g). And distance (in micrometres) of presynaptic sites to the closest DCV sorted by the value for DAN-i1 right and left together. Most of the presynaptic sites have a DCV associated within 1 μm.

Extended Data Figure 9 KC-to-MBON synaptic connections.

a, Percentage of mature KCs that are presynaptic to a given MBON. b, Frequency of the percentage of KCs presynaptic to MBONs (bin width is 10%). c, Frequency of the percentage of MBONs each KC connects to for single-claw and multi-claw KCs separately. All single-claw KCs connect with at least 75% of all MBONs present in their own hemisphere. d, Percentage of dendritic MBON inputs from individual KCs in the left brain hemisphere. KCs are ranked by their number of synapses to the MBON for each MBON separately (each line represents an MBON). Note the rank order of KCs is different for every MBON. Note also that a few MBONs receive very strong synaptic input from approximately 20 KCs and less than 3% from the remaining KCs, while most MBONs receive less than 4% of dendritic input from all KCs. e, Same as d, but for the right brain hemisphere. f, Effective strength of thermosensory input to MBONs. The thermosensory fraction is defined as the number of synapses received by an MBON from thermosensory KCs divided by 0.05 times the number of synapses received from all other KCs. The fraction thus represents the relative influence of an input that activates the thermosensory KCs compared with that of a typical stimulus that activates 5% of KCs.

Extended Data Figure 10 MBIN-to-KC synaptic connections.

a, Percentage of mature KCs that are postsynaptic to a given MBIN. On average, 68% of KCs in a compartment are postsynaptic to at least one MBIN of that compartment. b, Frequency of the percentage of KCs postsynaptic to MBINs (bin width is 10%). c, Frequency of the percentage of MBINs presynaptic to each KC for single-claw and multi-claw KCs separately. All single-claw KCs receive synaptic input from at least 50% of all MBINs present in their own hemisphere. d, Percentage of axonic outputs from MBINs connecting to individual KCs in the left brain hemisphere. KCs are ranked by their number of synapses they receive from an MBIN for each MBIN separately (each line represents an MBIN). Note the rank order of KCs is different for every MBIN. Note also that a few MBINs connect very strongly to approximately ten KCs and less than 2% to the remaining KCs, while most MBINs dedicate less than 2% of their axonic output to all KCs. e, Same as d, but for the right brain hemisphere.

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Supplementary Information

This file contains Supplementary Tables 2-7, Atlas Figures 1-2 and Supplementary References. (PDF 12081 kb)

Reporting Summary (PDF 94 kb)

Supplementary Table 1

This file contains the Connectivity matrix of the entire MB network. Neurons in rows are presynaptic to neurons in columns. PN-PN connections are almost all dendro-dendritic and occur within the antennal lobe. (ZIP 20 kb)

Supplementary Data

This file contains anatomical reconstruction data. (ZIP 14971 kb)

Supplementary Data

This file contains source data for the Main and Extended Data Figures. (ZIP 19905 kb)

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Eichler, K., Li, F., Litwin-Kumar, A. et al. The complete connectome of a learning and memory centre in an insect brain. Nature 548, 175–182 (2017). https://doi.org/10.1038/nature23455

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