[en] Neuronal firing sequences are thought to be the building blocks of information and broadcasting within the brain. Yet, it remains unclear when these sequences emerge during neurodevelopment. Here we demonstrate that structured firing sequences appear in spontaneous activity of human and murine brain organoids, both unguided and forebrain identity directed, as well as ex vivo neonatal murine cortical slices. We observed temporally rigid and flexible firing patterns in human and murine brain organoids and early postnatal murine somatosensory cortex, but not in dissociated primary cortical cultures. These results suggest that temporal sequences do not arise in an experience-dependent manner, but are rather constrained by a preconfigured architecture established during neurodevelopment. By demonstrating the developmental recapitulation of neural firing patterns, these findings highlight the potential of brain organoids as a model for neuronal circuit assembly.
Disciplines :
Life sciences: Multidisciplinary, general & others
Author, co-author :
van der Molen, Tjitse ; Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA ; Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA, USA ; Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA
Spaeth, Alex ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA ; Department of Electrical and Computer Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA
Chini, Mattia ; Université de Liège - ULiège > Département des sciences cliniques > Neurologie ; Institute of Developmental Neuroscience, Center for Molecular Neurobiology, Hamburg Center of Neuroscience, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Hernandez, Sebastian; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA ; Department of Electrical and Computer Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA
Kaurala, Gregory A; Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA
Schweiger, Hunter E; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA ; Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA
Duncan, Cole; Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA
McKenna, Sawyer; Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA
Geng, Jinghui ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA ; Department of Electrical and Computer Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA
Lim, Max; Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA, USA ; Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA
Bartram, Julian ; Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
Gänswein, Tobias ; Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
Dendukuri, Aditya; Department of Computer Science, University of California, Santa Barbara, Santa Barbara, CA, USA
Zhang, Zongren; Department of Physics, University of California, Santa Barbara, Santa Barbara, CA, USA
Gonzalez-Ferrer, Jesus; Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA
Bhaskaran-Nair, Kiran; Department of Biology, Washington University in St. Louis, St. Louis, MO, USA
Morson, Aidan L; Department of Physics, University of California, Santa Cruz, Santa Cruz, CA, USA
Harder, Cole R K; Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA
Petzold, Linda R; Department of Computer Science, University of California, Santa Barbara, Santa Barbara, CA, USA
Alam El Din, Dowlette-Mary; Center for Alternatives to Animal Testing (CAAT), Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
Laird, Jason; Center for Alternatives to Animal Testing (CAAT), Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
Schenke, Maren ; Center for Alternatives to Animal Testing (CAAT), Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
Smirnova, Lena ; Center for Alternatives to Animal Testing (CAAT), Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
Colquitt, Bradley M ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA ; Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA ; Institute for the Biology of Stem Cells, University of California, Santa Cruz, Santa Cruz, CA, USA
Mostajo-Radji, Mohammed A; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA
Hansma, Paul K; Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA, USA ; Department of Physics, University of California, Santa Barbara, Santa Barbara, CA, USA
Teodorescu, Mircea ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA ; Department of Electrical and Computer Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA
Hierlemann, Andreas ; Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
Hengen, Keith B ; Department of Biology, Washington University in St. Louis, St. Louis, MO, USA
Hanganu-Opatz, Ileana L; Institute of Developmental Neuroscience, Center for Molecular Neurobiology, Hamburg Center of Neuroscience, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Kosik, Kenneth S ; Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA, USA ; Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA
Sharf, Tal ; Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA. tsharf@ucsc.edu ; UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA, USA. tsharf@ucsc.edu ; Institute for the Biology of Stem Cells, University of California, Santa Cruz, Santa Cruz, CA, USA. tsharf@ucsc.edu
We would like to thank the members of the Braingeneers consortium for helpful discussions and D. Haussler for insightful comments. We would also like to thank members of the UC Santa Cruz Genomics Institute for helping with computing resources, in particular D. Parks for assistance with archiving the neurophysiology data. This study was supported by the National Science Foundation (NSF) Emerging Frontiers in Research and Innovation under award (NSF 2515389 to T.S.), UC Santa Cruz Baskin Engineering Seed Grant (to T.S.), Schmidt Futures Foundation (SF857 to M.T.), National Human Genome Research Institute under award (1RM1HG011543 to M.T.), German Research Foundation FOR5159 TP1 (437610067 to I.L.H.-O.), European Research Council advanced grant \u2018neuroXscales\u2019 (694829 to A.H.), Swiss NSF project (205320_188910/1 to A.H.), National Institutes of Health (NIH; T32 ES007141 to D.-M.A.E.D.) and International Foundation for Ethical Research (to D.-M.A.E.D.), Hopkins Discovery and Johns Hopkins SURPASS (to L.S.), John Douglas French Alzheimer\u2019s Foundation (to K.S.K.), NIH BRAIN Initiative (R01NS118442 to K.B.H.) and National Institute of Mental Health grant (1U24MH132628 to M.A.M.-R.). Through the National Research Platform, this work was supported in part by NSF awards (CNS-1730158, ACI-1540112, ACI-1541349, OAC-1826967, OAC-2112167, CNS-2100237 and CNS-2120019), the University of California Office of the President and the University of California San Diego\u2019s California Institute for Telecommunications and Information Technology/Qualcomm Institute.
Llinás RR Paré D Of dreaming and wakefulness Neuroscience 1991 44 521 535 1754050
Song S Sjöström PJ Reigl M Nelson S Chklovskii DB Highly nonrandom features of synaptic connectivity in local cortical circuits PLoS Biol. 2005 3 15737062 1054880 e68
Buzsáki G Mizuseki K The log-dynamic brain: how skewed distributions affect network operations Nat. Rev. Neurosci. 2014 15 264 278 24569488 4051294
Luczak A Barthó P Harris KD Spontaneous events outline the realm of possible sensory responses in neocortical populations Neuron 2009 62 413 425 19447096 2696272
Hemberger M Shein-Idelson M Pammer L Laurent G Reliable sequential activation of neural assemblies by single pyramidal cells in a three-layered cortex Neuron 2019 104 353 369 31439429
Riquelme JL Hemberger M Laurent G Gjorgjieva J Single spikes drive sequential propagation and routing of activity in a cortical network eLife 2023 12 36780217 9925052 e79928
Luczak A McNaughton BL Harris KD Packet-based communication in the cortex Nat. Rev. Neurosci. 2015 16 745 755 26507295
Pastalkova E Itskov V Amarasingham A Buzsáki G Internally generated cell assembly sequences in the rat hippocampus Science 2008 321 1322 1327 18772431 2570043
Dragoi G Tonegawa S Preplay of future place cell sequences by hippocampal cellular assemblies Nature 2011 469 397 401 21179088
Grosmark AD Buzsáki G Diversity in neural firing dynamics supports both rigid and learned hippocampal sequences Science 2016 351 1440 1443 27013730 4919122
Farooq U Dragoi G Emergence of preconfigured and plastic time-compressed sequences in early postnatal development Science 2019 363 168 173 30630930 6794005
Carrillo-Reid L Miller J-EK Hamm JP Jackson J Yuste R Endogenous sequential cortical activity evoked by visual stimuli J. Neurosci. 2015 35 8813 8828 26063915 4461687
Vaz AP Wittig JH Inati SK Zaghloul KA Replay of cortical spiking sequences during human memory retrieval Science 2020 367 1131 1134 32139543 7211396
Vaz AP Wittig JH Inati SK Zaghloul KA Backbone spiking sequence as a basis for preplay, replay, and default states in human cortex Nat. Commun. 2023 14 37550285 10406814 4723
Xie W et al. Neuronal sequences in population bursts encode information in human cortex Nature 2024 635 935 942 39415012 12279075
Huszár R Zhang Y Blockus H Buzsáki G Preconfigured dynamics in the hippocampus are guided by embryonic birthdate and rate of neurogenesis Nat. Neurosci. 2022 25 1201 1212 35995878 10807234
Eiraku M et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals Cell Stem Cell 2008 3 519 532 18983967
Lancaster MA et al. Cerebral organoids model human brain development and microcephaly Nature 2013 501 373 379 23995685
Birey F et al. Assembly of functionally integrated human forebrain spheroids Nature 2017 545 54 59 28445465 5805137
Quadrato G et al. Cell diversity and network dynamics in photosensitive human brain organoids Nature 2017 545 48 53 28445462 5659341
Giandomenico SL et al. Cerebral organoids at the air–liquid interface generate diverse nerve tracts with functional output Nat. Neurosci. 2019 22 669 679 30886407 6436729
Sharf T et al. Functional neuronal circuitry and oscillatory dynamics in human brain organoids Nat. Commun. 2022 13 35906223 9338020 4403
Qian X et al. Sliced human cortical organoids for modeling distinct cortical layer formation Cell Stem Cell 2020 26 766 781 32142682 7366517
Wang Y et al. Modeling human telencephalic development and autism-associated SHANK3 deficiency using organoids generated from single neural rosettes Nat. Commun. 2022 13 36202854 9537523 5688
Trujillo CA et al. Complex oscillatory waves emerging from cortical organoids model early human brain network development Cell Stem Cell 2019 25 558 569 31474560 6778040
Samarasinghe RA et al. Identification of neural oscillations and epileptiform changes in human brain organoids Nat. Neurosci. 2021 24 1488 1500 34426698 9070733
Alam El Din D-M et al. Human neural organoid microphysiological systems show the building blocks necessary for basic learning and memory Commun. Biol. 2025 8 40819006 12357958 1237
Yuan X et al. Versatile live-cell activity analysis platform for characterization of neuronal dynamics at single-cell and network level Nat. Commun. 2020 11 32978383 7519655 4854
Bartram J et al. Parallel reconstruction of the excitatory and inhibitory inputs received by single neurons reveals the synaptic basis of recurrent spiking. eLife 2024 12 RP86820
Koch C Rapp M Segev I A brief history of time (constants) Cereb. Cortex 1996 6 93 101 8670642
Maimon G Assad JA Beyond Poisson: increased spike-time regularity across primate parietal cortex Neuron 2009 62 426 440 19447097 2743683
Ma Z Turrigiano GG Wessel R Hengen KB Cortical circuit dynamics are homeostatically tuned to criticality in vivo Neuron 2019 104 655 664 31601510 6934140
Wilson KG The renormalization group: critical phenomena and the Kondo problem Rev. Mod. Phys. 1975 47 773 840
Dragoi G The generative grammar of the brain: a critique of internally generated representations Nat. Rev. Neurosci. 2024 25 60 75 38036709
Derdikman D Hildesheim R Ahissar E Arieli A Grinvald A Imaging spatiotemporal dynamics of surround inhibition in the barrels somatosensory cortex J. Neurosci. 2003 23 3100 3105 12716915 6742298
Murray JD et al. A hierarchy of intrinsic timescales across primate cortex Nat. Neurosci. 2014 17 1661 1663 25383900 4241138
Chini M Pfeffer T Hanganu-Opatz I An increase of inhibition drives the developmental decorrelation of neural activity eLife 2022 11 35975980 9448324 e78811
Okun M et al. Diverse coupling of neurons to populations in sensory cortex Nature 2015 521 511 515 25849776 4449271
Lynn CW Holmes CM Bialek W Schwab DJ Decomposing the local arrow of time in interacting systems Phys. Rev. Lett. 2022 129 118101 36154397 9751844
Chen Y-N Kostka JK Bitzenhofer SH Hanganu-Opatz IL Olfactory bulb activity shapes the development of entorhinal-hippocampal coupling and associated cognitive abilities Curr. Biol. 2023 33 4353 4366 37729915 10617757
Mochizuki Y et al. Similarity in neuronal firing regimes across mammalian species J. Neurosci. 2016 36 5736 5747 27225764 4879195
O’Byrne J Jerbi K How critical is brain criticality? Trends Neurosci. 2022 45 820 837 36096888
Xu Y Schneider A Wessel R Hengen KB Sleep restores an optimal computational regime in cortical networks Nat. Neurosci. 2024 27 328 338 38182837 11272063
Hopfield JJ Neural networks and physical systems with emergent collective computational abilities Proc. Natl Acad. Sci. USA 1982 79 2554 2558 6953413 346238
Kenet T Bibitchkov D Tsodyks M Grinvald A Arieli A Spontaneously emerging cortical representations of visual attributes Nature 2003 425 954 956 14586468
Han F Caporale N Dan Y Reverberation of recent visual experience in spontaneous cortical waves Neuron 2008 60 321 327 18957223 3576032
Cai H et al. Brain organoid reservoir computing for artificial intelligence Nat. Electron. 2023 6 1032 1039
Kanton S et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development Nature 2019 574 418 422 31619793
Chini M Hnida M Kostka JK Chen Y-N Hanganu-Opatz IL Preconfigured architecture of the developing mouse brain Cell Rep. 2024 43 38795344 114267
Kalemaki K et al. The developmental changes in intrinsic and synaptic properties of prefrontal neurons enhance local network activity from the second to the third postnatal weeks in mice Cereb. Cortex 2022 32 3633 3650 34905772
Che A et al. Layer I interneurons sharpen sensory maps during neonatal development Neuron 2018 99 98 116 29937280 6152945
Kirmse K et al. GABA depolarizes immature neurons and inhibits network activity in the neonatal neocortex in vivo Nat. Commun. 2015 6 26177896 7750
Roth JG et al. Spatially controlled construction of assembloids using bioprinting Nat. Commun. 2023 14 37468483 10356773 4346
Osaki T et al. Complex activity and short-term plasticity of human cerebral organoids reciprocally connected with axons Nat. Commun. 2024 15 38600094 11006899 2945
Carmena JM et al. Learning to control a brain–machine interface for reaching and grasping by primates PLoS Biol. 2003 1 14624244 261882 e42
Barabási DL Schuhknecht GFP Engert F Functional neuronal circuits emerge in the absence of developmental activity Nat. Commun. 2024 15 38191595 10774424 364
Kant, I. Critique of Pure Reason (Cambridge University Press, 1998).
Birtele M et al. Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis Nat. Neurosci. 2023 26 2090 2103 37946050 11349286
Birtele M Lancaster M Quadrato G Modelling human brain development and disease with organoids Nat. Rev. Mol. Cell Biol. 2025 26 389 412 39668188
Pollen AA et al. Establishing cerebral organoids as models of human-specific brain evolution Cell 2019 176 743 756 30735633 6544371
Fiddes IT et al. Human-specific NOTCH2NL genes affect notch signaling and cortical neurogenesis Cell 2018 173 1356 1369 29856954 5986104
Benito-Kwiecinski S et al. An early cell shape transition drives evolutionary expansion of the human forebrain Cell 2021 184 2084 2102 33765444 8054913
Buccino AP et al. SpikeInterface, a unified framework for spike sorting eLife 2020 9 e61834 33170122 7704107
Pachitariu, M., Steinmetz, N., Kadir, S., Carandini, M. & Harris, K. Fast and accurate spike sorting of high-channel count probes with KiloSort. In Proc. Advances in Neural Information Processing Systems (eds Lee, D. et al.) 4455–4463 (Curran Associates, Inc., 2016).
Hill DN Mehta SB Kleinfeld D Quality metrics to accompany spike sorting of extracellular signals J. Neurosci. 2011 31 8699 8705 21677152 3123734
Romero JC et al. Oligodendrogenesis and myelination tracing in a CRISPR/Cas9-engineered brain microphysiological system Front. Cell. Neurosci. 2023 16 36744062 9893511 1094291
Bartram J et al. Cortical up states induce the selective weakening of subthreshold synaptic inputs Nat. Commun. 2017 8 28939859 5610171 665
Holt GR Softky WR Koch C Douglas RJ Comparison of discharge variability in vitro and in vivo in cat visual cortex neurons J. Neurophysiol. 1996 75 1806 1814 8734581
Okun M et al. Population rate dynamics and multineuron firing patterns in sensory cortex J. Neurosci. 2012 32 17108 17119 23197704 3520056
Bates D Mächler M Zurich E Bolker BM Walker SC Fitting linear mixed-effects models using lme4 J. Stat. Softw. 2015 67 1 48
Kuznetsova A Brockhoff PB Christensen RHB lmerTest package: tests in linear mixed effects models J. Stat. Softw. 2017 82 1 26
Lüdecke D Ben-Shachar M Patil I Waggoner P Makowski D Performance: an R package for assessment, comparison and testing of statistical models J. Open Source Softw. 2021 6 3139
Long JA jtools: analysis and presentation of social scientific data J. Open Source Softw. 2024 9 6610
