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34 Publications

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    05/09/24 | Neurotransmitter classification from electron microscopy images at synaptic sites in Drosophila melanogaster
    Eckstein N, Bates AS, Champion A, Du M, Yin Y, Schlegel P, Lu AK, Rymer T, Finley-May S, Paterson T, Parekh R, Dorkenwald S, Matsliah A, Yu S, McKellar C, Sterling A, Eichler K, Costa M, Seung S, Murthy M, Hartenstein V, Jefferis GS, Funke J
    Cell. 2024 May 09;187(10):2574-2594.e23. doi: 10.1016/j.cell.2024.03.016

    High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.

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    04/22/24 | A Bayesian Solution to Count the Number of Molecules within a Diffraction Limited Spot
    Alexander Hillsley , Johannes Stein , Paul W. Tillberg , David L. Stern , Jan Funke
    bioRxiv. 2024 Apr 22:. doi: 10.1101/2024.04.18.590066

    We address the problem of inferring the number of independently blinking fluorescent light emitters, when only their combined intensity contributions can be observed at each timepoint. This problem occurs regularly in light microscopy of objects that are smaller than the diffraction limit, where one wishes to count the number of fluorescently labelled subunits. Our proposed solution directly models the photo-physics of the system, as well as the blinking kinetics of the fluorescent emitters as a fully differentiable hidden Markov model. Given a trace of intensity over time, our model jointly estimates the parameters of the intensity distribution per emitter, their blinking rates, as well as a posterior distribution of the total number of fluorescent emitters. We show that our model is consistently more accurate and increases the range of countable subunits by a factor of two compared to current state-of-the-art methods, which count based on autocorrelation and blinking frequency, Further-more, we demonstrate that our model can be used to investigate the effect of blinking kinetics on counting ability, and therefore can inform experimental conditions that will maximize counting accuracy.

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    12/12/23 | Model-Based Inference of Synaptic Plasticity Rules
    Yash Mehta , Danil Tyulmankov , Adithya E. Rajagopalan , Glenn C. Turner , James E. Fitzgerald , Jan Funke
    bioRxiv. 2023 Dec 12:. doi: 10.1101/2023.12.11.571103

    Understanding learning through synaptic plasticity rules in the brain is a grand challenge for neuroscience. Here we introduce a novel computational framework for inferring plasticity rules from experimental data on neural activity trajectories and behavioral learning dynamics. Our methodology parameterizes the plasticity function to provide theoretical interpretability and facilitate gradient-based optimization. For instance, we use Taylor series expansions or multilayer perceptrons to approximate plasticity rules, and we adjust their parameters via gradient descent over entire trajectories to closely match observed neural activity and behavioral data. Notably, our approach can learn intricate rules that induce long nonlinear time-dependencies, such as those incorporating postsynaptic activity and current synaptic weights. We validate our method through simulations, accurately recovering established rules, like Oja’s, as well as more complex hypothetical rules incorporating reward-modulated terms. We assess the resilience of our technique to noise and, as a tangible application, apply it to behavioral data from Drosophila during a probabilistic reward-learning experiment. Remarkably, we identify an active forgetting component of reward learning in flies that enhances the predictive accuracy of previous models. Overall, our modeling framework provides an exciting new avenue to elucidate the computational principles governing synaptic plasticity and learning in the brain.

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    10/26/23 | Neural-circuit basis of song preference learning in fruit flies
    Keisuke Imoto , Yuki Ishikawa , Yoshinori Aso , Jan Funke , Ryoya Tanaka , Azusa Kamikouchi
    bioRxiv. 2023 Oct 26:. doi: 10.1101/2023.10.24.563693

    As observed in human language learning and song learning in birds, the fruit fly Drosophila melanogaster changes its' auditory behaviors according to prior sound experiences. Female flies that have heard male courtship songs of the same species are less responsive to courtship songs of different species. This phenomenon, known as song preference learning in flies, requires GABAergic input to pC1 neurons in the central brain, with these neurons playing a key role in mating behavior by integrating multimodal sensory and internal information. The neural circuit basis of this GABAergic input, however, has not yet been identified. Here, we find that pCd-2 neurons, totaling four cells per hemibrain and expressing the sex-determination gene doublesex, provide the GABAergic input to pC1 neurons for song preference learning. First, RNAi-mediated knockdown of GABA production in pCd-2 neurons abolished song preference learning. Second, pCd-2 neurons directly, and in many cases mutually, connect with pC1 neurons, suggesting the existence of reciprocal circuits between pC1 and pCd-2 neurons. Finally, GABAergic and dopaminergic inputs to pCd-2 neurons are necessary for song preference learning. Together, this study suggests that reciprocal circuits between pC1 and pCd-2 neurons serve as a sensory and internal state-integrated hub, allowing flexible control over female copulation. Consequently, this provides a neural circuit model that underlies experience-dependent auditory plasticity.

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    10/01/23 | Unsupervised Learning of Object-Centric Embeddings for Cell Instance Segmentation in Microscopy Images
    Wolf S, Lalit M, McDole K, Funke J
    2023 IEEE/CVF International Conference on Computer Vision (ICCV). 2023 Oct 01:. doi: 10.1109/ICCV51070.2023.01944

    Segmentation of objects in microscopy images is required for many biomedical applications. We introduce object-centric embeddings (OCEs), which embed image patches such that the spatial offsets between patches cropped from the same object are preserved. Those learnt embeddings can be used to delineate individual objects and thus obtain instance segmentations. Here, we show theoretically that, under assumptions commonly found in microscopy images, OCEs can be learnt through a self-supervised task that predicts the spatial offset between image patches. Together, this forms an unsupervised cell instance segmentation method which we evaluate on nine diverse large-scale microscopy datasets. Segmentations obtained with our method lead to substantially improved results, compared to state-of-the-art baselines on six out of nine datasets, and perform on par on the remaining three datasets. If ground-truth annotations are available, our method serves as an excellent starting point for supervised training, reducing the required amount of ground-truth needed by one order of magnitude, thus substantially increasing the practical applicability of our method. Source code is available at

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    07/29/23 | Network Statistics of the Whole-Brain Connectome of Drosophila
    Albert Lin , Runzhe Yang , Sven Dorkenwald , Arie Matsliah , Amy R. Sterling , Philipp Schlegel , Szi-chieh Yu , Claire E. McKellar , Marta Costa , Katharina Eichler , Alexander Shakeel Bates , Nils Eckstein , Jan Funke , Gregory S.X.E. Jefferis , Mala Murthy
    bioRxiv. 2023 Jul 29:. doi: 10.1101/2023.07.29.551086

    Animal brains are complex organs composed of thousands of interconnected neurons. Characterizing the network properties of these brains is a requisite step towards understanding mechanisms of computation and information flow. With the completion of the Flywire project, we now have access to the connectome of a complete adult Drosophila brain, containing 130,000 neurons and millions of connections. Here, we present a statistical summary and data products of the Flywire connectome, delving into its network properties and topological features. To gain insights into local connectivity, we computed the prevalence of two- and three-node network motifs, examined their strengths and neurotransmitter compositions, and compared these topological metrics with wiring diagrams of other animals. We uncovered a population of highly connected neurons known as the “rich club” and identified subsets of neurons that may serve as integrators or broadcasters of signals. Finally, we examined subnetworks based on 78 anatomically defined brain regions. The freely available data and neuron populations presented here will serve as a foundation for models and experiments exploring the relationship between neural activity and anatomical structure.

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    07/22/23 | Towards Generalizable Organelle Segmentation in Volume Electron Microscopy.
    Heinrich L, Patton W, Bennett D, Ackerman D, Park G, Bogovic JA, Eckstein N, Petruncio A, Clements J, Pang S, Shan Xu C, Funke J, Korff W, Hess H, Lippincott-Schwartz J, Saalfeld S, Weigel A, CellMap Project Team
    Microscopy and Microanalysis. 2023 Jul 22;29(Supplement_1):975. doi: 10.1093/micmic/ozad067.487
    06/29/23 | Neuronal wiring diagram of an adult brain.
    Dorkenwald S, Matsliah A, Sterling AR, Schlegel P, Yu S, McKellar CE, Lin A, Costa M, Eichler K, Yin Y, Silversmith W, Schneider-Mizell C, Jordan CS, Brittain D, Halageri A, Kuehner K, Ogedengbe O, Morey R, Gager J, Kruk K, Perlman E, Yang R, Deutsch D, Bland D, Sorek M, Lu R, Macrina T, Lee K, Bae JA, Mu S, Nehoran B, Mitchell E, Popovych S, Wu J, Jia Z, Castro M, Kemnitz N, Ih D, Bates AS, Eckstein N, Funke J, Collman F, Bock DD, Jefferis GS, Seung HS, Murthy M, FlyWire Consortium
    bioRxiv. 2023 Jun 29:. doi: 10.1101/2023.06.27.546656

    Connections between neurons can be mapped by acquiring and analyzing electron microscopic (EM) brain images. In recent years, this approach has been applied to chunks of brains to reconstruct local connectivity maps that are highly informative, yet inadequate for understanding brain function more globally. Here, we present the first neuronal wiring diagram of a whole adult brain, containing 5×10 chemical synapses between ∼130,000 neurons reconstructed from a female . The resource also incorporates annotations of cell classes and types, nerves, hemilineages, and predictions of neurotransmitter identities. Data products are available by download, programmatic access, and interactive browsing and made interoperable with other fly data resources. We show how to derive a projectome, a map of projections between regions, from the connectome. We demonstrate the tracing of synaptic pathways and the analysis of information flow from inputs (sensory and ascending neurons) to outputs (motor, endocrine, and descending neurons), across both hemispheres, and between the central brain and the optic lobes. Tracing from a subset of photoreceptors all the way to descending motor pathways illustrates how structure can uncover putative circuit mechanisms underlying sensorimotor behaviors. The technologies and open ecosystem of the FlyWire Consortium set the stage for future large-scale connectome projects in other species.

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    06/06/23 | A Connectome of the Male Drosophila Ventral Nerve Cord
    Shin-ya Takemura , Kenneth J Hayworth , Gary B Huang , Michal Januszewski , Zhiyuan Lu , Elizabeth C Marin , Stephan Preibisch , C Shan Xu , John Bogovic , Andrew S Champion , Han S J Cheong , Marta Costa , Katharina Eichler , William Katz , Christopher Knecht , Feng Li , Billy J Morris , Christopher Ordish , Patricia K Rivlin , Philipp Schlegel , Kazunori Shinomiya , Tomke Sturner , Ting Zhao , Griffin Badalamente , Dennis Bailey , Paul Brooks , Brandon S Canino , Jody Clements , Michael Cook , Octave Duclos , Christopher R Dunne , Kelli Fairbanks , Siqi Fang , Samantha Finley-May , Audrey Francis , Reed George , Marina Gkantia , Kyle Harrington , Gary Patrick Hopkins , Joseph Hsu , Philip M Hubbard , Alexandre Javier , Dagmar Kainmueller , Wyatt Korff , Julie Kovalyak , Dominik Krzeminski , Shirley A Lauchie , Alanna Lohff , Charli Maldonado , Emily A Manley , Caroline Mooney , Erika Neace , Matthew Nichols , Omotara Ogundeyi , Nneoma Okeoma , Tyler Paterson , Elliott Phillips , Emily M Phillips , Caitlin Ribeiro , Sean M Ryan , Jon Thomson Rymer , Anne K Scott , Ashley L Scott , David Shepherd , Aya Shinomiya , Claire Smith , Alia Suleiman , Satoko Takemura , Iris Talebi , Imaan F M Tamimi , Eric T Trautman , Lowell Umayam , John J Walsh , Tansy Yang , Gerald M Rubin , Louis K Scheffer , Jan Funke , Stephan Saalfeld , Harald F Hess , Stephen M Plaza , Gwyneth M Card , Gregory S X E Jefferis , Stuart Berg
    bioRxiv. 2023 Jun 06:. doi: 10.1101/2023.06.05.543757

    Animal behavior is principally expressed through neural control of muscles. Therefore understanding how the brain controls behavior requires mapping neuronal circuits all the way to motor neurons. We have previously established technology to collect large-volume electron microscopy data sets of neural tissue and fully reconstruct the morphology of the neurons and their chemical synaptic connections throughout the volume. Using these tools we generated a dense wiring diagram, or connectome, for a large portion of the Drosophila central brain. However, in most animals, including the fly, the majority of motor neurons are located outside the brain in a neural center closer to the body, i.e. the mammalian spinal cord or insect ventral nerve cord (VNC). In this paper, we extend our effort to map full neural circuits for behavior by generating a connectome of the VNC of a male fly.

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    05/02/23 | A leaky integrate-and-fire computational model based on the connectome of the entire adult Drosophila brain reveals insights into sensorimotor processing
    Philip K. Shiu , Gabriella R. Sterne , Nico Spiller , Romain Franconville , Andrea Sandoval , Joie Zhou , Neha Simha , Chan Hyuk Kang , Seongbong Yu , Jinseop S. Kim , Sven Dorkenwald , Arie Matsliah , Philipp Schlegel , Szi-chieh Yu , Claire E. McKellar , Amy Sterling , Marta Costa , Katharina Eichler , Gregory S.X.E. Jefferis , Mala Murthy , Alexander Shakeel Bates , Nils Eckstein , Jan Funke , Salil S. Bidaye , Stefanie Hampel , Andrew M. Seeds , Kristin Scott
    bioRxiv. 2023 May 02:. doi: 10.1101/2023.05.02.539144

    The forthcoming assembly of the adult Drosophila melanogaster central brain connectome, containing over 125,000 neurons and 50 million synaptic connections, provides a template for examining sensory processing throughout the brain. Here, we create a leaky integrate-and-fire computational model of the entire Drosophila brain, based on neural connectivity and neurotransmitter identity, to study circuit properties of feeding and grooming behaviors. We show that activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation. Computational activation of neurons in the feeding region of the Drosophila brain predicts those that elicit motor neuron firing, a testable hypothesis that we validate by optogenetic activation and behavioral studies. Moreover, computational activation of different classes of gustatory neurons makes accurate predictions of how multiple taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. Our computational model predicts that the sugar and water pathways form a partially shared appetitive feeding initiation pathway, which our calcium imaging and behavioral experiments confirm. Additionally, we applied this model to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit that do not overlap with gustatory circuits, and accurately describes the circuit response upon activation of different mechanosensory subtypes. Our results demonstrate that modeling brain circuits purely from connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can accurately describe complete sensorimotor transformations.

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