From b756b524c96ed16f4aa3b761e0d1eb334371a214 Mon Sep 17 00:00:00 2001 From: cdpaiva Date: Tue, 7 Oct 2025 09:47:52 -0400 Subject: [PATCH 01/21] chore: add gh action for generating paper draft --- .github/workflows/draft-pdf.yaml | 24 ++++++++++++++++++++++++ 1 file changed, 24 insertions(+) create mode 100644 .github/workflows/draft-pdf.yaml diff --git a/.github/workflows/draft-pdf.yaml b/.github/workflows/draft-pdf.yaml new file mode 100644 index 0000000..f9bb6c4 --- /dev/null +++ b/.github/workflows/draft-pdf.yaml @@ -0,0 +1,24 @@ +name: Draft PDF +on: + push: + paths: + - paper/** + - .github/workflows/draft-pdf.yaml + +jobs: + paper: + runs-on: ubuntu-latest + name: Paper Draft + steps: + - name: Checkout + uses: actions/checkout@v4 + - name: Build draft PDF + uses: openjournals/openjournals-draft-action@master + with: + journal: joss + paper-path: paper/paper.md + - name: Upload + uses: actions/upload-artifact@v4 + with: + name: paper + path: paper/paper.pdf \ No newline at end of file From fd4dc84621b9f1d8390ca5dda09a9136caf4e2c6 Mon Sep 17 00:00:00 2001 From: cdpaiva Date: Tue, 7 Oct 2025 09:48:17 -0400 Subject: [PATCH 02/21] docs: add paper --- paper/figures/snazzy-fig1.png | Bin 0 -> 126332 bytes paper/figures/snazzy-fig2.png | Bin 0 -> 98471 bytes paper/figures/snazzy-fig3.png | Bin 0 -> 23785 bytes paper/figures/snazzy-fig4.png | Bin 0 -> 29541 bytes paper/paper.bib | 208 ++++++++++++++++++++++++++++++++++ paper/paper.md | 171 ++++++++++++++++++++++++++++ 6 files changed, 379 insertions(+) create mode 100644 paper/figures/snazzy-fig1.png create mode 100644 paper/figures/snazzy-fig2.png create mode 100644 paper/figures/snazzy-fig3.png create mode 100644 paper/figures/snazzy-fig4.png create mode 100644 paper/paper.bib create mode 100644 paper/paper.md diff --git a/paper/figures/snazzy-fig1.png b/paper/figures/snazzy-fig1.png new file mode 100644 index 0000000000000000000000000000000000000000..312bbacc543e38920628efa7524339da67ae2780 GIT binary patch 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z&(Ie)7T+Erb2}WRT<-6=pZaP{6k(ztOCT19`k-<8k{Qhp^l#d}5JW`LKvc3>*D$8b zBsPuOihSi(7pu#dxO03!oSl&Xh^JC6SoL}LiLs$Exzveii3VCmT>{gCn|ZmVKo=4U z`0Xbdl{4>d09MuLQG9_D8t}RD5|)-nQP&%`w^8q$;|FWD-rWc!Zm4qQPCExI;MSBh z?|SIl7Jug>u75ykkR$&ZLEKKnQ-Lov(_&nSTiTA)rbCQ6_kDHv>nxkM(mowRN0u#M znaUL^u6clCy=tjmn*z{+-t|!;3Xp*!UA$Vfs{Wre2~_Hn(fVBY;RREh zKa8CXT%xH_>31$**BaPaQD`V+t@Ac)B8rr*A1(cVcqg=A$H<>MIua1qT<7%l zh$UV|do%dyzpqGesGrcL|Mz&_vP*U()4bdc`j3QX6qsK>2F0Q~k$}wQm4v8_NU4yH G@BaaYb=)lg literal 0 HcmV?d00001 diff --git a/paper/paper.bib b/paper/paper.bib new file mode 100644 index 0000000..31a4f25 --- /dev/null +++ b/paper/paper.bib @@ -0,0 +1,208 @@ +@article{wu:2024, + title={Network state transitions during cortical development}, + ISSN={1471-003X}, + url={http://dx.doi.org/10.1038/s41583-024-00824-y}, + DOI={10.1038/s41583-024-00824-y}, + abstractNote={Mammalian cortical networks are active before synaptogenesis begins in earnest, before neuronal migration is complete, and well before an animal opens its eyes and begins to actively explore its surroundings. This early activity undergoes several transformations during development. The most important of these is a transition from episodic synchronous network events, which are necessary for patterning the neocortex into functionally related modules, to desynchronized activity that is computationally more powerful and efficient. Network desynchronization is perhaps the most dramatic and abrupt developmental event in an otherwise slow and gradual process of brain maturation. In this Review, we summarize what is known about the phenomenology of developmental synchronous activity in the rodent neocortex and speculate on the mechanisms that drive its eventual desynchronization. We argue that desynchronization of network activity is a fundamental step through which the cortex transitions from passive, bottom-up detection of sensory stimuli to active sensory processing with top-down modulation.}, + journal={Nature reviews. Neuroscience}, + author={Wu, Michelle W. and Kourdougli, Nazim and Portera-Cailliau, Carlos}, + year={2024}, + month=may, + language={en} } + +@article{akin:2020, + title={Activity regulates brain development in the fly}, + volume={65}, + ISSN={0959-437X}, + abstractNote={It has long been appreciated that activity sculpts the formation of neuronal circuits in the mammalian brain. By contrast, in Drosophila it was generally thought that genetically hardwired mechanisms of development, mediated by cell recognition molecules, are sufficient to specify the connectome-the complex and stereotyped pattern of connections between neurons. Here, we consider recent findings that activity during development also contributes to circuit assembly in the fly. These observations suggest that activity is a fundamental aspect of brain development in general. They also highlight the opportunities to learn about the role of this phenomenon through the extensive Drosophila toolkit for examining circuit development, structure, and function.}, + journal={Current opinion in genetics & development}, + publisher={Elsevier BV}, + author={Akin, Orkun and Zipursky, S. Lawrence}, + year={2020}, + month=dec, + pages={8–13}, + language={en} } + +@article{ardiel:2022, + title={Stereotyped behavioral maturation and rhythmic quiescence in C. elegans embryos}, + volume={11}, + ISSN={2050-084X}, + url={http://dx.doi.org/10.7554/eLife.76836}, + DOI={10.7554/eLife.76836}, + abstractNote={Systematic analysis of rich behavioral recordings is being used to uncover how circuits encode complex behaviors. Here, we apply this approach to embryos. What are the first embryonic behaviors and how do they evolve as early neurodevelopment ensues? To address these questions, we present a systematic description of behavioral maturation for Caenorhabditis elegans embryos. Posture libraries were built using a genetically encoded motion capture suit imaged with light-sheet microscopy and annotated using custom tracking software. Analysis of cell trajectories, postures, and behavioral motifs revealed a stereotyped developmental progression. Early movement is dominated by flipping between dorsal and ventral coiling, which gradually slows into a period of reduced motility. Late-stage embryos exhibit sinusoidal waves of dorsoventral bends, prolonged bouts of directed motion, and a rhythmic pattern of pausing, which we designate slow wave twitch (SWT). Synaptic transmission is required for late-stage motion but not for early flipping nor the intervening inactive phase. A high-throughput behavioral assay and calcium imaging revealed that SWT is elicited by the rhythmic activity of a quiescence-promoting neuron (RIS). Similar periodic quiescent states are seen prenatally in diverse animals and may play an important role in promoting normal developmental outcomes.}, + journal={eLife}, + publisher={eLife Sciences Publications, Ltd}, + author={Ardiel, Evan L. and Lauziere, Andrew and Xu, Stephen and Harvey, Brandon J. and Christensen, Ryan Patrick and Nurrish, Stephen and Kaplan, Joshua M. and Shroff, Hari}, + year={2022}, + month=aug, + keywords={C. elegans; behavior; embryo; neuropeptides; neuroscience}, + language={en} } + +@article{avasthi:2023, + title={Gotta catch ‘em all: Agar microchambers for high-throughput single-cell live imaging}, + ISSN={2998-4084}, + url={http://dx.doi.org/10.57844/arcadia-v1bg-6b60}, + DOI={10.57844/ARCADIA-V1BG-6B60}, + abstractNote={As part of an ongoing effort at Arcadia to enable high-throughput image-based comparisons, we need adaptable ways to capture and record the behavior of motile single-celled organisms across a diverse range of species. In a recent pub, we designed microchambers to observe motile cells, including algae and ciliates [1]. These 3D-printed microchambers used Teflon-coated wells paired with a microscope slide and a polymer coverslip to constrain cells within the field of view (FOV). While our original designs were useful in some contexts, we wanted to generalize our design to allow 1) more flexibility in chamber size and shape to accommodate different-sized organisms and better organism tracking, 2) the ability to capture single cells or organisms within chambers, 3) construction of the microchambers with fewer parts (easier to make chambers robustly!), and 4) to avoid the need for a 3D printer so that researchers can make chambers without costly machinery. We turned to inexpensive (~$200–300) custom-fabricated PDMS stamps to mold agar along with standard microscope slides and coverslips. We demonstrate several different use cases for agar microchambers, including 1) organismal tracking, 2) high-speed sub-cellular imaging, and 3) long-term imaging of unicellular organisms and nematode development. Finally, we provide a testable framework for choosing an ideal microchamber design based on organismal size. We hope that this resource will be useful for anyone interested in increasing throughput for phenotyping microorganisms with microscopy.}, + publisher={Arcadia Science}, + author={Avasthi, Prachee and Essock-Burns, Tara and Garcia, Galo, III and Gehring, Jase and Matus, David Q. and Mets, David G. and York, Ryan}, + year={2023}, + month=apr } + +@article{blankenship:2009, + title={Mechanisms underlying spontaneous patterned activity in developing neural circuits}, + volume={11}, + ISSN={1471-003X}, + abstractNote={Patterned, spontaneous activity occurs in many developing neural circuits, including the retina, the cochlea, the spinal cord, the cerebellum and the hippocampus, where it provides signals that are important for the development of neurons and their connections. Despite there being differences in adult architecture and output across these various circuits, the patterns of spontaneous network activity and the mechanisms that generate it are remarkably similar. The mechanisms can include a depolarizing action of GABA (gamma-aminobutyric acid), transient synaptic connections, extrasynaptic transmission, gap junction coupling and the presence of pacemaker-like neurons. Interestingly, spontaneous activity is robust; if one element of a circuit is disrupted another will generate similar activity. This research suggests that developing neural circuits exhibit transient and tunable features that maintain a source of correlated activity during crucial stages of development.}, + number={1}, + journal={Nature reviews. Neuroscience}, + publisher={Nature Publishing Group}, + author={Blankenship, Aaron G. and Feller, Marla B.}, + year={2009}, + pages={18–29} } + +@article{carreira-Rosario:2021, + title={Mechanosensory input during circuit formation shapes Drosophila motor behavior through patterned spontaneous network activity}, + volume={31}, + ISSN={0960-9822}, + abstractNote={Neural activity sculpts circuit wiring in many animals. In vertebrates, patterned spontaneous network activity (PaSNA) generates sensory maps and establishes local circuits.1-3 However, it remains unclear how PaSNA might shape neuronal circuits and behavior in invertebrates. Previous work in the developing Drosophila embryo discovered intrinsic muscle activity that did not require synaptic transmission, and hence was myogenic, preceding PaSNA.4-6 These studies, however, monitored muscle movement, not neural activity, and were therefore unable to observe how myogenic activity might relate to subsequent neural network engagement. Here we use calcium imaging to directly record neural activity and characterize the emergence of PaSNA. We demonstrate that the spatiotemporal properties of PaSNA are highly stereotyped across embryos, arguing for genetic programming. Neural activity begins well before it becomes patterned, emerging during the myogenic stage. Remarkably, inhibition of mechanosensory input, as well as inhibition of muscle contractions, results in premature and excessive PaSNA, demonstrating that muscle movement serves as a brake on this process. Finally, transient mechanosensory inhibition during PaSNA, followed by quantitative modeling of larval behavior, shows that mechanosensory modulation during development is required for proper larval foraging. This work provides a foundation for using the Drosophila embryo to study the role of PaSNA in circuit formation, provides mechanistic insight into how PaSNA is entrained by motor activity, and demonstrates that spontaneous network activity is essential for locomotor behavior. These studies argue that sensory feedback during the earliest stages of circuit formation can sculpt locomotor behaviors through innate motor learning.}, + number={23}, + journal={Current biology: CB}, + author={Carreira-Rosario, Arnaldo and York, Ryan A. and Choi, Minseung and Doe, Chris Q. and Clandinin, Thomas R.}, + year={2021}, + month=dec, + pages={5341–5349.e4}, + keywords={Drosophila embryo; behavioral development; development of locomotor behavior; nervous system development; neural circuit wiring; spontaneous network activity}, + language={en} } + +@article{crisp:2008, + title={The development of motor coordination in Drosophila embryos}, + volume={3717}, + author={Crisp, Sarah and Evers, Jan Felix and Fiala, André and Bate, Michael}, + year={2008}, + pages={3707–3717}, + keywords={coordination; drosophila; embryo; movement; muscle} } + +@article{donoughe:2018, + title={High-throughput live-imaging of embryos in microwell arrays using a modular specimen mounting system}, + volume={7}, + ISSN={2046-6390}, + abstractNote={High-throughput live-imaging of embryos is an essential technique in developmental biology, but it is difficult and costly to mount and image embryos in consistent conditions. Here, we present OMMAwell, a simple, reusable device to easily mount dozens of embryos in arrays of agarose microwells with customizable dimensions and spacing. OMMAwell can be configured to mount specimens for upright or inverted microscopes, and includes a reservoir to hold live-imaging medium to maintain constant moisture and osmolarity of specimens during time-lapse imaging. All device components can be fabricated by cutting pieces from a sheet of acrylic using a laser cutter or by making them with a 3D printer. We demonstrate how to design a custom mold and use it to live-image dozens of embryos at a time. We include descriptions, schematics, and design files for 13 additional molds for nine animal species, including most major traditional laboratory models and a number of emerging model systems. Finally, we provide instructions for researchers to customize OMMAwell inserts for embryos or tissues not described herein.}, + number={7}, + journal={Biology open}, + publisher={The Company of Biologists}, + author={Donoughe, Seth and Kim, Chiyoung and Extavour, Cassandra G.}, + year={2018}, + month=jul, + pages={bio031260}, + keywords={Development; Embryogenesis; High-throughput; Image analysis; Microscopy; Quantitative imaging; Time lapse}, + language={en} } + +@article{fischler:1981, + title={Random sample consensus: a paradigm for model fitting with applications to image analysis and automated cartography}, + volume={24}, + ISSN={0001-0782}, + abstractNote={A new paradigm, Random Sample Consensus (RANSAC), for fitting a model to experimental data is introduced. RANSAC is capable of interpreting/smoothing data containing a significant percentage of gross errors, and is thus ideally suited for applications in automated image analysis where interpretation is based on the data provided by error-prone feature detectors. A major portion of this paper describes the application of RANSAC to the Location Determination Problem (LDP): Given an image depicting a set of landmarks with known locations, determine that point in space from which the image was obtained. In response to a RANSAC requirement, new results are derived on the minimum number of landmarks needed to obtain a solution, and algorithms are presented for computing these minimum-landmark solutions in closed form. These results provide the basis for an automatic system that can solve the LDP under difficult viewing}, + number={6}, + journal={Communications of the ACM}, + publisher={Association for Computing Machinery (ACM)}, + author={Fischler, Martin A. and Bolles, Robert C.}, + year={1981}, + month=jun, + pages={381–395}, + language={en} } + +@article{giovannucci:2019, + title={CaImAn an open source tool for scalable calcium imaging data analysis}, + volume={8}, + ISSN={2050-084X}, + url={http://dx.doi.org/10.7554/eLife.38173}, + DOI={10.7554/eLife.38173}, + abstractNote={Advances in fluorescence microscopy enable monitoring larger brain areas in-vivo with finer time resolution. The resulting data rates require reproducible analysis pipelines that are reliable, fully automated, and scalable to datasets generated over the course of months. We present CaImAn, an open-source library for calcium imaging data analysis. CaImAn provides automatic and scalable methods to address problems common to pre-processing, including motion correction, neural activity identification, and registration across different sessions of data collection. It does this while requiring minimal user intervention, with good scalability on computers ranging from laptops to high-performance computing clusters. CaImAn is suitable for two-photon and one-photon imaging, and also enables real-time analysis on streaming data. To benchmark the performance of CaImAn we collected and combined a corpus of manual annotations from multiple labelers on nine mouse two-photon datasets. We demonstrate that CaImAn achieves near-human performance in detecting locations of active neurons.}, + journal={eLife}, + publisher={eLife Sciences Publications, Ltd}, + author={Giovannucci, Andrea and Friedrich, Johannes and Gunn, Pat and Kalfon, Jérémie and Brown, Brandon L. and Koay, Sue Ann and Taxidis, Jiannis and Najafi, Farzaneh and Gauthier, Jeffrey L. and Zhou, Pengcheng and Khakh, Baljit S. and Tank, David W. and Chklovskii, Dmitri B. and Pnevmatikakis, Eftychios A.}, + year={2019}, + month=jan, + keywords={calcium imaging; data analysis; mouse; neuroscience; one-photon; open source; software; two-photon; zebrafish}, + language={en} } + +@article{lin:2016, + title={Genetically encoded indicators of neuronal activity}, + volume={19}, + ISSN={1097-6256}, + abstractNote={Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.}, + number={9}, + journal={Nature neuroscience}, + publisher={Nature Publishing Group}, + author={Lin, Michael Z. and Schnitzer, Mark J.}, + year={2016}, + month=aug, + pages={1142–1153}, + language={en} } + +@unpublished{menzies:2024, + title={A microRNA that controls the emergence of embryonic movement}, + url={http://dx.doi.org/10.7554/eLife.95209.2}, + DOI={10.7554/elife.95209.2}, + abstractNote={Summary Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here we investigate the genetic basis underlying the embryonic onset of movement in Drosophila focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, we first develop a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos, and using this system, discover that the Drosophila miRNA miR-2b-1 plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging we define that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests we identify a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene – which we term Movement Modulator (Motor) – as a genetic target that mechanistically links miR-2b-1 to the onset of movement. Cell-specific genetic reconstitution of miR-2b-1 expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that miR-2b-1 affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. Our work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in Drosophila as well as in other species.}, + journal={eLife}, + author={Menzies, Jonathan A. C. and Chagas, Andre M. and Baden, Tom and Alonso, Claudio R.}, + year={2024}, + month=jun, + language={en} } + +@article{nakai:2001, + title={A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein}, + volume={19}, + ISSN={1087-0156}, + abstractNote={Recently, several groups have developed green fluorescent protein (GFP)-based Ca(2+) probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca(2+) probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K(d) for Ca(2+) of 235 nM. Association kinetics of Ca(2+) binding were faster at higher Ca(2+) concentrations, with time constants decreasing from 230 ms at 0.2 microM Ca(2+) to 2.5 ms at 1 microM Ca(2+). Dissociation kinetics (tau approximately 200 ms) are independent of Ca(2+) concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.}, + number={2}, + journal={Nature biotechnology}, + publisher={Springer Science and Business Media LLC}, + author={Nakai, J. and Ohkura, M. and Imoto, K.}, + year={2001}, + month=feb, + pages={137–141}, + language={en} } + +@article{otsu:1979, + title={A threshold selection method from gray-level histograms}, + volume={9}, + ISSN={0018-9472}, + abstractNote={IEEE Xplore, delivering full text access to the world’s highest quality technical literature in engineering and technology. | IEEE Xplore}, + number={1}, + journal={IEEE transactions on systems, man, and cybernetics}, + publisher={Institute of Electrical and Electronics Engineers (IEEE)}, + author={Otsu, Nobuyuki}, + year={1979}, + month=jan, + pages={62–66}, + language={en} } + +@unpublished{pachitariu:2016, + title={Suite2p: beyond 10,000 neurons with standard two-photon microscopy}, + url={https://www.biorxiv.org/content/10.1101/061507v2.abstract}, + DOI={10.1101/061507}, + abstractNote={AbstractTwo-photon microscopy of calcium-dependent sensors has enabled unprecedented recordings from vast populations of neurons. While the sensors and microscopes have matured over several generations of development, computational methods to process the resulting movies remain inefficient and can give results that are hard to interpret. Here we introduce Suite2p: a fast, accurate and complete pipeline that registers raw movies, detects active cells, extracts their calcium traces and infers their spike times. Suite2p runs on standard workstations, operates faster than real time, and recovers ~2 times more cells than the previous state-of-the-art method. Its low computational load allows routine detection of ~10,000 cells simultaneously with standard two-photon resonant-scanning microscopes. Recordings at this scale promise to reveal the fine structure of activity in large populations of neurons or large populations of subcellular structures such as synaptic boutons.}, + journal={bioRxiv}, + publisher={bioRxiv}, + author={Pachitariu, Marius and Stringer, Carsen and Dipoppa, Mario and Schröder, Sylvia and Rossi, L. Federico and Dalgleish, Henry and Carandini, Matteo and Harris, Kenneth D.}, + year={2016}, + month=jun, + pages={061507}, + language={en} } + +@article{virtanen:2020, + title={SciPy 1.0: fundamental algorithms for scientific computing in Python}, + volume={17}, + ISSN={1548-7091}, + abstractNote={SciPy is an open-source scientific computing library for the Python programming language. Since its initial release in 2001, SciPy has become a de facto standard for leveraging scientific algorithms in Python, with over 600 unique code contributors, thousands of dependent packages, over 100,000 dependent repositories and millions of downloads per year. In this work, we provide an overview of the capabilities and development practices of SciPy 1.0 and highlight some recent technical developments.}, + number={3}, + journal={Nature methods}, + publisher={Springer Science and Business Media LLC}, + author={Virtanen, Pauli and Gommers, Ralf and Oliphant, Travis E. and Haberland, Matt and Reddy, Tyler and Cournapeau, David and Burovski, Evgeni and Peterson, Pearu and Weckesser, Warren and Bright, Jonathan and van der Walt, Stéfan J. and Brett, Matthew and Wilson, Joshua and Millman, K. Jarrod and Mayorov, Nikolay and Nelson, Andrew R. J. and Jones, Eric and Kern, Robert and Larson, Eric and Carey, C. J. and Polat, İlhan and Feng, Yu and Moore, Eric W. and VanderPlas, Jake and Laxalde, Denis and Perktold, Josef and Cimrman, Robert and Henriksen, Ian and Quintero, E. A. and Harris, Charles R. and Archibald, Anne M. and Ribeiro, Antônio H. and Pedregosa, Fabian and van Mulbregt, Paul and SciPy 1.0 Contributors}, + year={2020}, + month=mar, + pages={261–272}, + language={en} } diff --git a/paper/paper.md b/paper/paper.md new file mode 100644 index 0000000..a34eb2c --- /dev/null +++ b/paper/paper.md @@ -0,0 +1,171 @@ +--- +title: "SNAzzy: an image processing pipeline for investigating global Synchronous Network Activity" +tags: + - Calcium imaging + - Widefield microscopy + - Image analysis + - Drosophila + - Spontaneous Neural Activity + - Neurodevelopment + - Circuit Wiring +authors: + - name: Carlos D. Paiva + orcid: 0009-0006-1836-7210 + affiliation: "1, 2" + - name: Alana J. Evora + orcid: 0009-0002-3174-7839 + affiliation: "1, 2" + - name: Shirui Zheng + orcid: 0009-0006-1836-7208 + affiliation: "1, 2" + - name: Arnaldo Carreira-Rosario + orcid: 0000-0003-0202-3858 + corresponding: true + affiliation: "1, 2" +affiliations: + - name: Department of Biology, Duke Unviersity, Durham, NC 27708 + index: 1 + - name: Department of Neurobiology, Duke Unviersity, Durham, NC 27708 + index: 2 +date: 6 October 2025 +bibliography: paper.bib +--- + +# Summary + +Genetically encoded fluorescent indicators are powerful tools for monitoring biological processes in live samples using light microscopy {@lin:2016, @nakai:2001}. +When combined with a large field of view, a single time-lapse recording has the potential to capture many specimens, facilitating high-throughput data collection. +However, the simultaneous recording of many biological samples across time points produces large, multidimensional datasets that are challenging to process and analyze. +We present `SNAzzy`, a Python package for studying synchronous network activity (SNA) in Drosophila embryos via high-throughput microscopy. +SNA is a hallmark of developing nervous systems (Wu, Kourdougli, and Portera-Cailliau 2024; Blankenship and Feller 2009; Akin and Zipursky 2020), often studied using genetically encoded calcium indicators to monitor neural activity in vivo. +`SNAzzy` processes and analyzes time-lapse datasets taken from live samples using fluorescent widefield microscopy. +Each dataset contains dozens of individual specimens in the same field of view and thousands of time points. +The software offers individual specimen cropping for optimization of storage and processing, adaptive regions of interest for quantification of fluorescence and changes in morphology over time, a custom peak detection algorithm, and a graphical user interface for data visualization, curation, and dataset comparison. +This tool can be readily applied to analyze fluorescent intensities in time-lapse microscopy experiments that involve simultaneous imaging of multiple samples, particularly small-sized specimens (Donoughe Kim, and Extavour 2018; Avasthi et al. 2023). + +# Statement of need + +During synchronous network activity (SNA), many neurons fire synchronously, generating waves of activity that span across large portions of the nervous system (Blankenship and Feller 2009; Wu, Kourdougli, and Portera-Cailliau 2024; Akin and Zipursky 2020). +In Drosophila embryos, SNA typically lasts 4 hours, during which the nervous system undergoes a stereotyped morphological change via ventral nerve cord condensation (Crisp et al 2008; Carreira-Rosario et al. 2021). +To gain an understanding of SNA, it is essential to quantify waves of activity in the nervous system while also tracking morphology as a proxy of neurodevelopment. +For these reasons, we combine a commonly used genetically encoded calcium indicator (GECI) that reflects neural activity with a structural fluorophore (Carreira-Rosario et al. +2021). +The structural fluorophore signal remains stable, independent of neural activity, making it suitable for continuous tracking morphology of the nerve cord. +To record many embryos during SNA, we use a wide-field fluorescence microscopy system that captures the GECI and structural fluorophore signal of dozens of developing embryos for over 5 hours. + +We were unable to find a tool designed for widefield microscopy that rapidly processes multiple specimens, quantifies levels of fluorophore activity, and incorporates a peak-finding algorithm suitable for global calcium traces. +`SNAzzy` is designed to investigate global levels of neural activity across multiple developing embryos simultaneously. + +## Tracking of multiple “adaptive ROIs” + +To the best of our knowledge, there are no other packages that provide functionality for automated parsing of raw images of many live specimens into activity and morphological quantifications. +Other studies have employed manual selection of regions of interest (ROIs) and used static ROIs (Menzies et al. 2024; Ardiel et al. 2022; Carreira-Rosario et al. 2021). +Manual selection often generates imprecise ROIs, which can lead to inaccurate quantifications, and is also cumbersome and prone to human error. +Static ROIs are not reliable for detecting the fluorescent signal of live specimens that change in morphology and move while imaging. +`SNAzzy` fills these gaps as an accessible pipeline for the automated analysis of multiple live samples in parallel. +The pipeline generates an “adaptive ROI” that changes frame-by-frame for each specimen. +This enables the accurate tracking of fluorescence intensity as well as changes in tissue morphology or size. +`SNAzzy`’s design provides an automated, modular, and fully auditable workflow, and ultimately contributes to more reproducible and comparable results across experiments. + +## Capturing global Calcium dynamics + +To the best of our knowledge, there are no open-source packages that provide tools for performing automated data analysis and quantification of global calcium dynamics. +Most open-source tools available for analyzing neuronal activity using GECI focus on segmenting individual neurons within a single specimen. +CaImAn (Giovannucci et al. 2019) and Suite2p (Pachitariu et al. 2016) are among the most widely used. +These packages detect calcium dynamics and use individual neuron statistics to perform spike inference, but do not offer direct peak detection on the calcium signal. +Furthermore, they are optimized for two-photon microscopy as opposed to wide-field microscopy. +`SNAzzy` provides a series of automated analyses and quantifications to analyze global calcium levels in time-series acquired with widefield microscopes. + +![Schematic of the SNAzzy pipeline. +Time-lapse taken from fluorescent widefield microscopes (raw data) enters the processing stage (green). +The processing stage outputs two types of CSV files: time series of signal intensities from each recorded channel and ROI length. +CSV files enter the analysis stage (blue) to generate normalized fluorescent traces and detect peaks along with other signal processing metrics. +These initial traces can be visualized to curate the data. +Curation generates a configuration file that works as metadata across platforms and users. +Curated data can be reanalyzed and used to visualize final data and compare across groups (yellow). +Analysis and output stages are performed in the GUI (red dashed box), along with other metrics. +Dashed arrows indicate optional steps.](figures/snazzy-fig1.png) + +# Pipeline Description + +The initial input for `SNAzzy` (Figure 1) is raw time-lapse imaging data containing multiple embryos. +Each embryo expresses a GECI (dynamic fluorophore) and a structural fluorophore. +Fluorophores are imaged in different optical channels. + +The first pipeline step converts the raw data to TIF format, thereby avoiding compatibility issues that may arise when parsing different proprietary formats (Figure 1). +All embryos are then segmented using histogram equalization followed by intensity threshold binarization (Otsu 1979). +Boxes surrounding the segments are cropped into individual time-lapses for each embryo. +Cropping results in a substantial memory reduction, as most background pixels are removed, with cropped images typically accounting for around 40% of the original size. + +The next step is to process each individual specimen. +First, the ROI, which in our case is the entire central nervous system (CNS), is defined by binarizing the structural channel and selecting the largest connected component. +This process is repeated at every time point to generate an “adaptive ROI”. +From these adaptive ROI, the average signal intensity for both channels is extracted. +The results are saved as CSV files and are the basis for downstream analysis. + +![ROI length measurement algorithm and validation. +A) Steps to calculate the ROI length. +The ROI length is calculated by estimating the centerline (red line) using points of maximum (dots) in the distance transform, followed by RANSAC to ignore outliers (orange dots). +B) Validation of the method as relative error (measured - annotated) / annotated. +Each whisker bar summarizes the relative error for frames taken at intervals of 50 timepoints. +C) Comparison of absolute values over a time series for three representative embryos.](figures/snazzy-fig2.png) + +The ROI is also used to measure the length of the CNS (Figure 2). +Drosophila embryo CNS length serves as an internal proxy for neurodevelopmental stages, enabling more accurate comparisons across embryos (Carreira-Rosario et al. 2021). +The CNS length is calculated by centerline estimation. +First, a distance transform is applied to the binarized image, and local maxima points are detected. +Depending on the embryo's orientation, some points may be part of the brain lobes and must be filtered out to accurately measure the CNS length. +To obtain a robust centerline estimate that can ignore outliers, we use RANSAC (Fischler and Bolles 1981) over the local maxima points and measure the overlap between the fitted line and the binary image. +CNS length is also detected frame by frame and exported as a CSV file (Figure 1). + +![Figure 3: Peak detection algorithm. +A low-pass filter (orange line) is applied to the ∆F/F signal (black line) to remove fast transients. +The peak in the filtered signal (orange dot) is then ported back to the ∆F/F (blue dot) signal by selecting the leftmost peak within a search window (blue lines).](figures/snazzy-fig3.png) + +The package utilizes average signal intensity measurements to calculate ∆F/F traces and peaks. +For ∆F/F, we first calculate the ratiometric signal (dynamic signal / structural signal) and then its baseline, which is defined as the average of the N lowest values within a sliding window. +The generated ∆F/F traces contain long-duration bouts of activity with superimposed fast transients (Figure 3). +The former represents the bursts of activity and is the most relevant for the initial analysis. +To mark only these more prolonged bouts, we apply a low-pass frequency filter to omit transients. +Peaks in the filtered trace are detected using SciPy (Virtanen et al. 2020). +Finally, the detected peaks are ported to the original ∆F/F signal. + +Results can be visualized and curated in a graphical user interface (GUI) implemented in `PyQt6` (Figure 4). +During curation, researchers can modify data analysis parameters, which are persisted in a JSON configuration file and utilized by the core analysis code across different machines and users. +Finally, a large number of different metrics derived from ∆F/F, CNS length, and peaks can be visualized and plotted using the GUI. + +![Figure 4: GUI for data validation, curation, visualization and plotting. +Initial GUI screen. +A ∆F/F trace (white) and the corresponding peaks (magenta dots) are shown. +The low-passed signal (green line) is used as a reference to determine peaks. +The GUI enables the modification of analysis parameters, visualization of data, and comparison of metrics across groups of experiments, as well as manual adjustment of peak data.](figures/snazzy-fig4.png) + +In conclusion, genetically encoded fluorescent indicators and microscopy systems are evolving rapidly, increasing the data acquisition throughput. +Custom open-source tools are needed to handle such large data files. +`SNAzzy` addresses this by offering an automated, scalable, and user-friendly platform for analyzing synchronous network activity in developing embryos. +As an open and versatile solution, `SNAzzy` offers tools for a broader range of applications in time-lapse fluorescence imaging across diverse biological systems. + +# Acknowledgments + +We acknowledge Newt PenkoffLidbeck and Berfin Azizoglu for feedback on the manuscript. +his work was partially supported by R00…. + +# References + +Akin, Orkun, and S. Lawrence Zipursky. 2020. “Activity Regulates Brain Development in the Fly.” Current Opinion in Genetics & Development 65 (December):8–13. +Ardiel, Evan L., Andrew Lauziere, Stephen Xu, Brandon J. Harvey, Ryan Patrick Christensen, Stephen Nurrish, Joshua M. Kaplan, and Hari Shroff. 2022. “Stereotyped Behavioral Maturation and Rhythmic Quiescence in C. Elegans Embryos.” eLife 11 (August). https://doi.org/10.7554/eLife.76836. +Avasthi, Prachee, Tara Essock-Burns, Galo Garcia III, Jase Gehring, David Q. Matus, David G. Mets, and Ryan York. 2023. “Gotta Catch ‘em All: Agar Microchambers for High-Throughput Single-Cell Live Imaging,” April. https://doi.org/10.57844/ARCADIA-V1BG-6B60. +Blankenship, Aaron G., and Marla B. Feller. 2009. “Mechanisms Underlying Spontaneous Patterned Activity in Developing Neural Circuits.” Nature Reviews. Neuroscience 11 (1): 18–29. +Carreira-Rosario, Arnaldo, Ryan A. York, Minseung Choi, Chris Q. Doe, and Thomas R. Clandinin. 2021. “Mechanosensory Input during Circuit Formation Shapes Drosophila Motor Behavior through Patterned Spontaneous Network Activity.” Current Biology: CB 31 (23): 5341–49.e4. +Crisp, Sarah, Jan Felix Evers, André Fiala, and Michael Bate. 2008. “The Development of Motor Coordination in Drosophila Embryos” 3717:3707–17. +Donoughe, Seth, Chiyoung Kim, and Cassandra G. Extavour. 2018. “High-Throughput Live-Imaging of Embryos in Microwell Arrays Using a Modular Specimen Mounting System.” Biology Open 7 (7): bio031260. +Fischler, Martin A., and Robert C. Bolles. 1981. “Random Sample Consensus: A Paradigm for Model Fitting with Applications to Image Analysis and Automated Cartography.” Communications of the ACM 24 (6): 381–95. +Giovannucci, Andrea, Johannes Friedrich, Pat Gunn, Jérémie Kalfon, Brandon L. Brown, Sue Ann Koay, Jiannis Taxidis, et al. 2019. “CaImAn an Open Source Tool for Scalable Calcium Imaging Data Analysis.” eLife 8 (January). https://doi.org/10.7554/eLife.38173. +Lin, Michael Z., and Mark J. Schnitzer. 2016. “Genetically Encoded Indicators of Neuronal Activity.” Nature Neuroscience 19 (9): 1142–53. +Menzies, Jonathan A. C., Andre M. Chagas, Tom Baden, and Claudio R. Alonso. 2024. “A microRNA That Controls the Emergence of Embryonic Movement.” eLife. https://doi.org/10.7554/elife.95209.2. +Nakai, J., M. Ohkura, and K. Imoto. 2001. “A High Signal-to-Noise Ca(2+) Probe Composed of a Single Green Fluorescent Protein.” Nature Biotechnology 19 (2): 137–41. +Otsu, Nobuyuki. 1979. “A Threshold Selection Method from Gray-Level Histograms.” IEEE Transactions on Systems, Man, and Cybernetics 9 (1): 62–66. +Pachitariu, Marius, Carsen Stringer, Mario Dipoppa, Sylvia Schröder, L. Federico Rossi, Henry Dalgleish, Matteo Carandini, and Kenneth D. Harris. 2016. “Suite2p: Beyond 10,000 Neurons with Standard Two-Photon Microscopy.” bioRxiv. bioRxiv. https://doi.org/10.1101/061507. +Virtanen, Pauli, Ralf Gommers, Travis E. Oliphant, Matt Haberland, Tyler Reddy, David Cournapeau, Evgeni Burovski, et al. 2020. “SciPy 1.0: Fundamental Algorithms for Scientific Computing in Python.” Nature Methods 17 (3): 261–72. +Wu, Michelle W., Nazim Kourdougli, and Carlos Portera-Cailliau. 2024. “Network State Transitions during Cortical Development.” Nature Reviews. Neuroscience, May. https://doi.org/10.1038/s41583-024-00824-y. + From 07600b4404188b0375803885b9f179185a94b270 Mon Sep 17 00:00:00 2001 From: cdpaiva Date: Wed, 8 Oct 2025 11:59:23 -0400 Subject: [PATCH 03/21] docs: fix md syntax --- paper/paper.bib | 17 +---------------- paper/paper.md | 49 ++++++++++++++++++++++++------------------------- 2 files changed, 25 insertions(+), 41 deletions(-) diff --git a/paper/paper.bib b/paper/paper.bib index 31a4f25..3460c50 100644 --- a/paper/paper.bib +++ b/paper/paper.bib @@ -3,7 +3,6 @@ @article{wu:2024 ISSN={1471-003X}, url={http://dx.doi.org/10.1038/s41583-024-00824-y}, DOI={10.1038/s41583-024-00824-y}, - abstractNote={Mammalian cortical networks are active before synaptogenesis begins in earnest, before neuronal migration is complete, and well before an animal opens its eyes and begins to actively explore its surroundings. This early activity undergoes several transformations during development. The most important of these is a transition from episodic synchronous network events, which are necessary for patterning the neocortex into functionally related modules, to desynchronized activity that is computationally more powerful and efficient. Network desynchronization is perhaps the most dramatic and abrupt developmental event in an otherwise slow and gradual process of brain maturation. In this Review, we summarize what is known about the phenomenology of developmental synchronous activity in the rodent neocortex and speculate on the mechanisms that drive its eventual desynchronization. We argue that desynchronization of network activity is a fundamental step through which the cortex transitions from passive, bottom-up detection of sensory stimuli to active sensory processing with top-down modulation.}, journal={Nature reviews. Neuroscience}, author={Wu, Michelle W. and Kourdougli, Nazim and Portera-Cailliau, Carlos}, year={2024}, @@ -14,7 +13,6 @@ @article{akin:2020 title={Activity regulates brain development in the fly}, volume={65}, ISSN={0959-437X}, - abstractNote={It has long been appreciated that activity sculpts the formation of neuronal circuits in the mammalian brain. By contrast, in Drosophila it was generally thought that genetically hardwired mechanisms of development, mediated by cell recognition molecules, are sufficient to specify the connectome-the complex and stereotyped pattern of connections between neurons. Here, we consider recent findings that activity during development also contributes to circuit assembly in the fly. These observations suggest that activity is a fundamental aspect of brain development in general. They also highlight the opportunities to learn about the role of this phenomenon through the extensive Drosophila toolkit for examining circuit development, structure, and function.}, journal={Current opinion in genetics & development}, publisher={Elsevier BV}, author={Akin, Orkun and Zipursky, S. Lawrence}, @@ -29,7 +27,6 @@ @article{ardiel:2022 ISSN={2050-084X}, url={http://dx.doi.org/10.7554/eLife.76836}, DOI={10.7554/eLife.76836}, - abstractNote={Systematic analysis of rich behavioral recordings is being used to uncover how circuits encode complex behaviors. Here, we apply this approach to embryos. What are the first embryonic behaviors and how do they evolve as early neurodevelopment ensues? To address these questions, we present a systematic description of behavioral maturation for Caenorhabditis elegans embryos. Posture libraries were built using a genetically encoded motion capture suit imaged with light-sheet microscopy and annotated using custom tracking software. Analysis of cell trajectories, postures, and behavioral motifs revealed a stereotyped developmental progression. Early movement is dominated by flipping between dorsal and ventral coiling, which gradually slows into a period of reduced motility. Late-stage embryos exhibit sinusoidal waves of dorsoventral bends, prolonged bouts of directed motion, and a rhythmic pattern of pausing, which we designate slow wave twitch (SWT). Synaptic transmission is required for late-stage motion but not for early flipping nor the intervening inactive phase. A high-throughput behavioral assay and calcium imaging revealed that SWT is elicited by the rhythmic activity of a quiescence-promoting neuron (RIS). Similar periodic quiescent states are seen prenatally in diverse animals and may play an important role in promoting normal developmental outcomes.}, journal={eLife}, publisher={eLife Sciences Publications, Ltd}, author={Ardiel, Evan L. and Lauziere, Andrew and Xu, Stephen and Harvey, Brandon J. and Christensen, Ryan Patrick and Nurrish, Stephen and Kaplan, Joshua M. and Shroff, Hari}, @@ -43,7 +40,6 @@ @article{avasthi:2023 ISSN={2998-4084}, url={http://dx.doi.org/10.57844/arcadia-v1bg-6b60}, DOI={10.57844/ARCADIA-V1BG-6B60}, - abstractNote={As part of an ongoing effort at Arcadia to enable high-throughput image-based comparisons, we need adaptable ways to capture and record the behavior of motile single-celled organisms across a diverse range of species. In a recent pub, we designed microchambers to observe motile cells, including algae and ciliates [1]. These 3D-printed microchambers used Teflon-coated wells paired with a microscope slide and a polymer coverslip to constrain cells within the field of view (FOV). While our original designs were useful in some contexts, we wanted to generalize our design to allow 1) more flexibility in chamber size and shape to accommodate different-sized organisms and better organism tracking, 2) the ability to capture single cells or organisms within chambers, 3) construction of the microchambers with fewer parts (easier to make chambers robustly!), and 4) to avoid the need for a 3D printer so that researchers can make chambers without costly machinery. We turned to inexpensive (~$200–300) custom-fabricated PDMS stamps to mold agar along with standard microscope slides and coverslips. We demonstrate several different use cases for agar microchambers, including 1) organismal tracking, 2) high-speed sub-cellular imaging, and 3) long-term imaging of unicellular organisms and nematode development. Finally, we provide a testable framework for choosing an ideal microchamber design based on organismal size. We hope that this resource will be useful for anyone interested in increasing throughput for phenotyping microorganisms with microscopy.}, publisher={Arcadia Science}, author={Avasthi, Prachee and Essock-Burns, Tara and Garcia, Galo, III and Gehring, Jase and Matus, David Q. and Mets, David G. and York, Ryan}, year={2023}, @@ -53,7 +49,6 @@ @article{blankenship:2009 title={Mechanisms underlying spontaneous patterned activity in developing neural circuits}, volume={11}, ISSN={1471-003X}, - abstractNote={Patterned, spontaneous activity occurs in many developing neural circuits, including the retina, the cochlea, the spinal cord, the cerebellum and the hippocampus, where it provides signals that are important for the development of neurons and their connections. Despite there being differences in adult architecture and output across these various circuits, the patterns of spontaneous network activity and the mechanisms that generate it are remarkably similar. The mechanisms can include a depolarizing action of GABA (gamma-aminobutyric acid), transient synaptic connections, extrasynaptic transmission, gap junction coupling and the presence of pacemaker-like neurons. Interestingly, spontaneous activity is robust; if one element of a circuit is disrupted another will generate similar activity. This research suggests that developing neural circuits exhibit transient and tunable features that maintain a source of correlated activity during crucial stages of development.}, number={1}, journal={Nature reviews. Neuroscience}, publisher={Nature Publishing Group}, @@ -61,11 +56,10 @@ @article{blankenship:2009 year={2009}, pages={18–29} } -@article{carreira-Rosario:2021, +@article{carreira:2021, title={Mechanosensory input during circuit formation shapes Drosophila motor behavior through patterned spontaneous network activity}, volume={31}, ISSN={0960-9822}, - abstractNote={Neural activity sculpts circuit wiring in many animals. In vertebrates, patterned spontaneous network activity (PaSNA) generates sensory maps and establishes local circuits.1-3 However, it remains unclear how PaSNA might shape neuronal circuits and behavior in invertebrates. Previous work in the developing Drosophila embryo discovered intrinsic muscle activity that did not require synaptic transmission, and hence was myogenic, preceding PaSNA.4-6 These studies, however, monitored muscle movement, not neural activity, and were therefore unable to observe how myogenic activity might relate to subsequent neural network engagement. Here we use calcium imaging to directly record neural activity and characterize the emergence of PaSNA. We demonstrate that the spatiotemporal properties of PaSNA are highly stereotyped across embryos, arguing for genetic programming. Neural activity begins well before it becomes patterned, emerging during the myogenic stage. Remarkably, inhibition of mechanosensory input, as well as inhibition of muscle contractions, results in premature and excessive PaSNA, demonstrating that muscle movement serves as a brake on this process. Finally, transient mechanosensory inhibition during PaSNA, followed by quantitative modeling of larval behavior, shows that mechanosensory modulation during development is required for proper larval foraging. This work provides a foundation for using the Drosophila embryo to study the role of PaSNA in circuit formation, provides mechanistic insight into how PaSNA is entrained by motor activity, and demonstrates that spontaneous network activity is essential for locomotor behavior. These studies argue that sensory feedback during the earliest stages of circuit formation can sculpt locomotor behaviors through innate motor learning.}, number={23}, journal={Current biology: CB}, author={Carreira-Rosario, Arnaldo and York, Ryan A. and Choi, Minseung and Doe, Chris Q. and Clandinin, Thomas R.}, @@ -87,7 +81,6 @@ @article{donoughe:2018 title={High-throughput live-imaging of embryos in microwell arrays using a modular specimen mounting system}, volume={7}, ISSN={2046-6390}, - abstractNote={High-throughput live-imaging of embryos is an essential technique in developmental biology, but it is difficult and costly to mount and image embryos in consistent conditions. Here, we present OMMAwell, a simple, reusable device to easily mount dozens of embryos in arrays of agarose microwells with customizable dimensions and spacing. OMMAwell can be configured to mount specimens for upright or inverted microscopes, and includes a reservoir to hold live-imaging medium to maintain constant moisture and osmolarity of specimens during time-lapse imaging. All device components can be fabricated by cutting pieces from a sheet of acrylic using a laser cutter or by making them with a 3D printer. We demonstrate how to design a custom mold and use it to live-image dozens of embryos at a time. We include descriptions, schematics, and design files for 13 additional molds for nine animal species, including most major traditional laboratory models and a number of emerging model systems. Finally, we provide instructions for researchers to customize OMMAwell inserts for embryos or tissues not described herein.}, number={7}, journal={Biology open}, publisher={The Company of Biologists}, @@ -102,7 +95,6 @@ @article{fischler:1981 title={Random sample consensus: a paradigm for model fitting with applications to image analysis and automated cartography}, volume={24}, ISSN={0001-0782}, - abstractNote={A new paradigm, Random Sample Consensus (RANSAC), for fitting a model to experimental data is introduced. RANSAC is capable of interpreting/smoothing data containing a significant percentage of gross errors, and is thus ideally suited for applications in automated image analysis where interpretation is based on the data provided by error-prone feature detectors. A major portion of this paper describes the application of RANSAC to the Location Determination Problem (LDP): Given an image depicting a set of landmarks with known locations, determine that point in space from which the image was obtained. In response to a RANSAC requirement, new results are derived on the minimum number of landmarks needed to obtain a solution, and algorithms are presented for computing these minimum-landmark solutions in closed form. These results provide the basis for an automatic system that can solve the LDP under difficult viewing}, number={6}, journal={Communications of the ACM}, publisher={Association for Computing Machinery (ACM)}, @@ -118,7 +110,6 @@ @article{giovannucci:2019 ISSN={2050-084X}, url={http://dx.doi.org/10.7554/eLife.38173}, DOI={10.7554/eLife.38173}, - abstractNote={Advances in fluorescence microscopy enable monitoring larger brain areas in-vivo with finer time resolution. The resulting data rates require reproducible analysis pipelines that are reliable, fully automated, and scalable to datasets generated over the course of months. We present CaImAn, an open-source library for calcium imaging data analysis. CaImAn provides automatic and scalable methods to address problems common to pre-processing, including motion correction, neural activity identification, and registration across different sessions of data collection. It does this while requiring minimal user intervention, with good scalability on computers ranging from laptops to high-performance computing clusters. CaImAn is suitable for two-photon and one-photon imaging, and also enables real-time analysis on streaming data. To benchmark the performance of CaImAn we collected and combined a corpus of manual annotations from multiple labelers on nine mouse two-photon datasets. We demonstrate that CaImAn achieves near-human performance in detecting locations of active neurons.}, journal={eLife}, publisher={eLife Sciences Publications, Ltd}, author={Giovannucci, Andrea and Friedrich, Johannes and Gunn, Pat and Kalfon, Jérémie and Brown, Brandon L. and Koay, Sue Ann and Taxidis, Jiannis and Najafi, Farzaneh and Gauthier, Jeffrey L. and Zhou, Pengcheng and Khakh, Baljit S. and Tank, David W. and Chklovskii, Dmitri B. and Pnevmatikakis, Eftychios A.}, @@ -131,7 +122,6 @@ @article{lin:2016 title={Genetically encoded indicators of neuronal activity}, volume={19}, ISSN={1097-6256}, - abstractNote={Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.}, number={9}, journal={Nature neuroscience}, publisher={Nature Publishing Group}, @@ -145,7 +135,6 @@ @unpublished{menzies:2024 title={A microRNA that controls the emergence of embryonic movement}, url={http://dx.doi.org/10.7554/eLife.95209.2}, DOI={10.7554/elife.95209.2}, - abstractNote={Summary Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here we investigate the genetic basis underlying the embryonic onset of movement in Drosophila focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, we first develop a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos, and using this system, discover that the Drosophila miRNA miR-2b-1 plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging we define that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests we identify a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene – which we term Movement Modulator (Motor) – as a genetic target that mechanistically links miR-2b-1 to the onset of movement. Cell-specific genetic reconstitution of miR-2b-1 expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that miR-2b-1 affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. Our work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in Drosophila as well as in other species.}, journal={eLife}, author={Menzies, Jonathan A. C. and Chagas, Andre M. and Baden, Tom and Alonso, Claudio R.}, year={2024}, @@ -156,7 +145,6 @@ @article{nakai:2001 title={A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein}, volume={19}, ISSN={1087-0156}, - abstractNote={Recently, several groups have developed green fluorescent protein (GFP)-based Ca(2+) probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca(2+) probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K(d) for Ca(2+) of 235 nM. Association kinetics of Ca(2+) binding were faster at higher Ca(2+) concentrations, with time constants decreasing from 230 ms at 0.2 microM Ca(2+) to 2.5 ms at 1 microM Ca(2+). Dissociation kinetics (tau approximately 200 ms) are independent of Ca(2+) concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.}, number={2}, journal={Nature biotechnology}, publisher={Springer Science and Business Media LLC}, @@ -170,7 +158,6 @@ @article{otsu:1979 title={A threshold selection method from gray-level histograms}, volume={9}, ISSN={0018-9472}, - abstractNote={IEEE Xplore, delivering full text access to the world’s highest quality technical literature in engineering and technology. | IEEE Xplore}, number={1}, journal={IEEE transactions on systems, man, and cybernetics}, publisher={Institute of Electrical and Electronics Engineers (IEEE)}, @@ -184,7 +171,6 @@ @unpublished{pachitariu:2016 title={Suite2p: beyond 10,000 neurons with standard two-photon microscopy}, url={https://www.biorxiv.org/content/10.1101/061507v2.abstract}, DOI={10.1101/061507}, - abstractNote={AbstractTwo-photon microscopy of calcium-dependent sensors has enabled unprecedented recordings from vast populations of neurons. While the sensors and microscopes have matured over several generations of development, computational methods to process the resulting movies remain inefficient and can give results that are hard to interpret. Here we introduce Suite2p: a fast, accurate and complete pipeline that registers raw movies, detects active cells, extracts their calcium traces and infers their spike times. Suite2p runs on standard workstations, operates faster than real time, and recovers ~2 times more cells than the previous state-of-the-art method. Its low computational load allows routine detection of ~10,000 cells simultaneously with standard two-photon resonant-scanning microscopes. Recordings at this scale promise to reveal the fine structure of activity in large populations of neurons or large populations of subcellular structures such as synaptic boutons.}, journal={bioRxiv}, publisher={bioRxiv}, author={Pachitariu, Marius and Stringer, Carsen and Dipoppa, Mario and Schröder, Sylvia and Rossi, L. Federico and Dalgleish, Henry and Carandini, Matteo and Harris, Kenneth D.}, @@ -197,7 +183,6 @@ @article{virtanen:2020 title={SciPy 1.0: fundamental algorithms for scientific computing in Python}, volume={17}, ISSN={1548-7091}, - abstractNote={SciPy is an open-source scientific computing library for the Python programming language. Since its initial release in 2001, SciPy has become a de facto standard for leveraging scientific algorithms in Python, with over 600 unique code contributors, thousands of dependent packages, over 100,000 dependent repositories and millions of downloads per year. In this work, we provide an overview of the capabilities and development practices of SciPy 1.0 and highlight some recent technical developments.}, number={3}, journal={Nature methods}, publisher={Springer Science and Business Media LLC}, diff --git a/paper/paper.md b/paper/paper.md index a34eb2c..7d516b9 100644 --- a/paper/paper.md +++ b/paper/paper.md @@ -10,7 +10,7 @@ tags: - Circuit Wiring authors: - name: Carlos D. Paiva - orcid: 0009-0006-1836-7210 + orcid: 0009-0007-6658-2620 affiliation: "1, 2" - name: Alana J. Evora orcid: 0009-0002-3174-7839 @@ -27,29 +27,28 @@ affiliations: index: 1 - name: Department of Neurobiology, Duke Unviersity, Durham, NC 27708 index: 2 -date: 6 October 2025 +date: 8 October 2025 bibliography: paper.bib --- # Summary -Genetically encoded fluorescent indicators are powerful tools for monitoring biological processes in live samples using light microscopy {@lin:2016, @nakai:2001}. +Genetically encoded fluorescent indicators are powerful tools for monitoring biological processes in live samples using light microscopy [@lin:2016; @nakai:2001]. When combined with a large field of view, a single time-lapse recording has the potential to capture many specimens, facilitating high-throughput data collection. However, the simultaneous recording of many biological samples across time points produces large, multidimensional datasets that are challenging to process and analyze. We present `SNAzzy`, a Python package for studying synchronous network activity (SNA) in Drosophila embryos via high-throughput microscopy. -SNA is a hallmark of developing nervous systems (Wu, Kourdougli, and Portera-Cailliau 2024; Blankenship and Feller 2009; Akin and Zipursky 2020), often studied using genetically encoded calcium indicators to monitor neural activity in vivo. +SNA is a hallmark of developing nervous systems [@wu:2024; @blankenship:2009; @akin:2020], often studied using genetically encoded calcium indicators to monitor neural activity in vivo. `SNAzzy` processes and analyzes time-lapse datasets taken from live samples using fluorescent widefield microscopy. Each dataset contains dozens of individual specimens in the same field of view and thousands of time points. The software offers individual specimen cropping for optimization of storage and processing, adaptive regions of interest for quantification of fluorescence and changes in morphology over time, a custom peak detection algorithm, and a graphical user interface for data visualization, curation, and dataset comparison. -This tool can be readily applied to analyze fluorescent intensities in time-lapse microscopy experiments that involve simultaneous imaging of multiple samples, particularly small-sized specimens (Donoughe Kim, and Extavour 2018; Avasthi et al. 2023). +This tool can be readily applied to analyze fluorescent intensities in time-lapse microscopy experiments that involve simultaneous imaging of multiple samples, particularly small-sized specimens [@donoughe:2018; @avasthi:2023]. # Statement of need -During synchronous network activity (SNA), many neurons fire synchronously, generating waves of activity that span across large portions of the nervous system (Blankenship and Feller 2009; Wu, Kourdougli, and Portera-Cailliau 2024; Akin and Zipursky 2020). -In Drosophila embryos, SNA typically lasts 4 hours, during which the nervous system undergoes a stereotyped morphological change via ventral nerve cord condensation (Crisp et al 2008; Carreira-Rosario et al. 2021). +During synchronous network activity (SNA), many neurons fire synchronously, generating waves of activity that span across large portions of the nervous system [@blankenship:2009; @wu:2024; @akin:2020] +In Drosophila embryos, SNA typically lasts 4 hours, during which the nervous system undergoes a stereotyped morphological change via ventral nerve cord condensation [@crisp:2008; @carreira:2021] To gain an understanding of SNA, it is essential to quantify waves of activity in the nervous system while also tracking morphology as a proxy of neurodevelopment. -For these reasons, we combine a commonly used genetically encoded calcium indicator (GECI) that reflects neural activity with a structural fluorophore (Carreira-Rosario et al. -2021). +For these reasons, we combine a commonly used genetically encoded calcium indicator (GECI) that reflects neural activity with a structural fluorophore [@carreira:2021]. The structural fluorophore signal remains stable, independent of neural activity, making it suitable for continuous tracking morphology of the nerve cord. To record many embryos during SNA, we use a wide-field fluorescence microscopy system that captures the GECI and structural fluorophore signal of dozens of developing embryos for over 5 hours. @@ -59,7 +58,7 @@ We were unable to find a tool designed for widefield microscopy that rapidly pro ## Tracking of multiple “adaptive ROIs” To the best of our knowledge, there are no other packages that provide functionality for automated parsing of raw images of many live specimens into activity and morphological quantifications. -Other studies have employed manual selection of regions of interest (ROIs) and used static ROIs (Menzies et al. 2024; Ardiel et al. 2022; Carreira-Rosario et al. 2021). +Other studies have employed manual selection of regions of interest (ROIs) and used static ROIs [@menzies:2024; @ardiel:2022; @carreira:2021]. Manual selection often generates imprecise ROIs, which can lead to inaccurate quantifications, and is also cumbersome and prone to human error. Static ROIs are not reliable for detecting the fluorescent signal of live specimens that change in morphology and move while imaging. `SNAzzy` fills these gaps as an accessible pipeline for the automated analysis of multiple live samples in parallel. @@ -71,7 +70,7 @@ This enables the accurate tracking of fluorescence intensity as well as changes To the best of our knowledge, there are no open-source packages that provide tools for performing automated data analysis and quantification of global calcium dynamics. Most open-source tools available for analyzing neuronal activity using GECI focus on segmenting individual neurons within a single specimen. -CaImAn (Giovannucci et al. 2019) and Suite2p (Pachitariu et al. 2016) are among the most widely used. +CaImAn [@giovannucci:2019], and Suite2p [@pachitariu:2016] are among the most widely used. These packages detect calcium dynamics and use individual neuron statistics to perform spike inference, but do not offer direct peak detection on the calcium signal. Furthermore, they are optimized for two-photon microscopy as opposed to wide-field microscopy. `SNAzzy` provides a series of automated analyses and quantifications to analyze global calcium levels in time-series acquired with widefield microscopes. @@ -84,16 +83,16 @@ These initial traces can be visualized to curate the data. Curation generates a configuration file that works as metadata across platforms and users. Curated data can be reanalyzed and used to visualize final data and compare across groups (yellow). Analysis and output stages are performed in the GUI (red dashed box), along with other metrics. -Dashed arrows indicate optional steps.](figures/snazzy-fig1.png) +Dashed arrows indicate optional steps.\label{fig:fig1}](figures/snazzy-fig1.png) # Pipeline Description -The initial input for `SNAzzy` (Figure 1) is raw time-lapse imaging data containing multiple embryos. +The initial input for `SNAzzy` \autoref{fig:fig1} is raw time-lapse imaging data containing multiple embryos. Each embryo expresses a GECI (dynamic fluorophore) and a structural fluorophore. Fluorophores are imaged in different optical channels. -The first pipeline step converts the raw data to TIF format, thereby avoiding compatibility issues that may arise when parsing different proprietary formats (Figure 1). -All embryos are then segmented using histogram equalization followed by intensity threshold binarization (Otsu 1979). +The first pipeline step converts the raw data to TIF format, thereby avoiding compatibility issues that may arise when parsing different proprietary formats \autoref{fig:fig1}. +All embryos are then segmented using histogram equalization followed by intensity threshold binarization [@otsu:1979]. Boxes surrounding the segments are cropped into individual time-lapses for each embryo. Cropping results in a substantial memory reduction, as most background pixels are removed, with cropped images typically accounting for around 40% of the original size. @@ -108,29 +107,29 @@ A) Steps to calculate the ROI length. The ROI length is calculated by estimating the centerline (red line) using points of maximum (dots) in the distance transform, followed by RANSAC to ignore outliers (orange dots). B) Validation of the method as relative error (measured - annotated) / annotated. Each whisker bar summarizes the relative error for frames taken at intervals of 50 timepoints. -C) Comparison of absolute values over a time series for three representative embryos.](figures/snazzy-fig2.png) +C) Comparison of absolute values over a time series for three representative embryos.\label{fig:fig2}](figures/snazzy-fig2.png) -The ROI is also used to measure the length of the CNS (Figure 2). -Drosophila embryo CNS length serves as an internal proxy for neurodevelopmental stages, enabling more accurate comparisons across embryos (Carreira-Rosario et al. 2021). +The ROI is also used to measure the length of the CNS \autoref{fig:fig2}. +Drosophila embryo CNS length serves as an internal proxy for neurodevelopmental stages, enabling more accurate comparisons across embryos [@carreira:2021]. The CNS length is calculated by centerline estimation. First, a distance transform is applied to the binarized image, and local maxima points are detected. Depending on the embryo's orientation, some points may be part of the brain lobes and must be filtered out to accurately measure the CNS length. -To obtain a robust centerline estimate that can ignore outliers, we use RANSAC (Fischler and Bolles 1981) over the local maxima points and measure the overlap between the fitted line and the binary image. -CNS length is also detected frame by frame and exported as a CSV file (Figure 1). +To obtain a robust centerline estimate that can ignore outliers, we use RANSAC [@fischler:1981] over the local maxima points and measure the overlap between the fitted line and the binary image. +CNS length is also detected frame by frame and exported as a CSV file \autoref{fig:fig1}. ![Figure 3: Peak detection algorithm. A low-pass filter (orange line) is applied to the ∆F/F signal (black line) to remove fast transients. -The peak in the filtered signal (orange dot) is then ported back to the ∆F/F (blue dot) signal by selecting the leftmost peak within a search window (blue lines).](figures/snazzy-fig3.png) +The peak in the filtered signal (orange dot) is then ported back to the ∆F/F (blue dot) signal by selecting the leftmost peak within a search window (blue lines).\label{fig:fig3}](figures/snazzy-fig3.png) The package utilizes average signal intensity measurements to calculate ∆F/F traces and peaks. For ∆F/F, we first calculate the ratiometric signal (dynamic signal / structural signal) and then its baseline, which is defined as the average of the N lowest values within a sliding window. -The generated ∆F/F traces contain long-duration bouts of activity with superimposed fast transients (Figure 3). +The generated ∆F/F traces contain long-duration bouts of activity with superimposed fast transients \autoref{fig:fig3}. The former represents the bursts of activity and is the most relevant for the initial analysis. To mark only these more prolonged bouts, we apply a low-pass frequency filter to omit transients. -Peaks in the filtered trace are detected using SciPy (Virtanen et al. 2020). +Peaks in the filtered trace are detected using SciPy [@virtanen:2020]. Finally, the detected peaks are ported to the original ∆F/F signal. -Results can be visualized and curated in a graphical user interface (GUI) implemented in `PyQt6` (Figure 4). +Results can be visualized and curated in a graphical user interface (GUI) implemented in `PyQt6` \autoref{fig:fig4}. During curation, researchers can modify data analysis parameters, which are persisted in a JSON configuration file and utilized by the core analysis code across different machines and users. Finally, a large number of different metrics derived from ∆F/F, CNS length, and peaks can be visualized and plotted using the GUI. @@ -138,7 +137,7 @@ Finally, a large number of different metrics derived from ∆F/F, CNS length, an Initial GUI screen. A ∆F/F trace (white) and the corresponding peaks (magenta dots) are shown. The low-passed signal (green line) is used as a reference to determine peaks. -The GUI enables the modification of analysis parameters, visualization of data, and comparison of metrics across groups of experiments, as well as manual adjustment of peak data.](figures/snazzy-fig4.png) +The GUI enables the modification of analysis parameters, visualization of data, and comparison of metrics across groups of experiments, as well as manual adjustment of peak data.\label{fig:fig4}](figures/snazzy-fig4.png) In conclusion, genetically encoded fluorescent indicators and microscopy systems are evolving rapidly, increasing the data acquisition throughput. Custom open-source tools are needed to handle such large data files. 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