Juergen A. Knoblich

Functional neural networks emerge as developing neurons form synaptic connections. Revealing how these connections are organized and perturbed in disease requires linking single-neuron connectivity to molecular state across thousands of networks, which is challenging with current methods. We developed COSCO (Connectomics by Sequencing in Cerebral Organoids), combining barcoded monosynaptic rabies tracing with single-cell RNA sequencing to reconstruct over 10,000 networks across organoids. We demonstrate that organoid circuitry self-organizes into stereotyped network motifs governed by cell-intrinsic wiring rules aligning with features of mouse cortical connectivity. Projecting network topology onto transcriptomic space reveals that wiring correlates with cell-adhesion and synaptic gene expression programs. In a Tuberous Sclerosis Complex model, pathological hyperexcitability coincides with topological rewiring: mutant networks become more heterogeneous, driven by the transcriptional distribution of upper layer excitatory cell states. This work establishes cerebral organoids as a model to study self-organization of human connectivity, and its disruption in neurodevelopmental disease. Abstract Figure:

The human brain is incomplete at birth, with streams of late-born interneurons migrating into the postnatal cortex. This process is absent in rodents, limiting mechanistic investigation to post-mortem tissue. Here, we show that human cerebral organoids cultured for over six months recapitulate postnatal interneuron migration, enabling its mechanistic and genetic dissection. Live imaging and mathematical modeling revealed that interneurons intrinsically migrate in chains, while astrocyte interactions promote stream organization. Spatial and single-cell transcriptomics identified a distinct molecular signature of human postnatal migratory streams enriched for neurodevelopmental disorder-associated genes. Perturbation of the autism-associated genes CNTN5 and FGF14 differentially disrupted chain organization and migration. Thus, long-term cerebral organoids enable mechanistic analysis of previously inaccessible aspects of postnatal human brain development and their contributions to neurodevelopmental disease.

The adult mammalian brain has limited regenerative capacity yet retains substantial potential for functional reorganization after experience, injury or disease1-3. While local plasticity at injury sites has been described4,5, the brain-wide consequences of a focal injury and the biological processes that drive them remain unknown. To address this, we performed an unbiased, whole brain screen of neuronal activity at single cell resolution following a focal injury to the primary visual cortex (V1) in mice. Olfactory processing areas emerged as loci of remote activation. We further show that V1 injury promotes the recruitment of adult born neurons into the olfactory bulb (OB) and potentiates cortical feedback onto bulbar circuits. Combining high-density multielectrode array recordings with two-photon calcium imaging revealed OB circuit refinement characterized by increased synchrony and sharpened tuning of principal output neurons. These circuit-level functional enhancements coincide with improved behavioral performance in odor-guided tasks. Motor cortex lesions do not elicit similar cellular and behavioral responses, suggesting that such distal adaptation only develops when injury is coupled with increased olfactory demand. Together, these data demonstrate that the adult brain can recruit alternative sensory circuits distant from a focal lesion to undergo adaptive, functionally relevant reorganization, and implicate adult neurogenesis as a contributing mechanism. Our findings expose a previously underappreciated degree of remote plasticity and reveal a novel role of adult neurogenesis in sensory compensation.

Summary: Tissue-clearing and light-sheet microscopy have transformed volumetric imaging of intact organs, yet limited mechanistic understanding of dehydration-based clearing continues to constrain rational protocol design and broader applicability. Here, we define the cardinal chemical and physical principles underlying dehydration-based tissue-clearing and establish a new pipeline for large-volume imaging. To maximize imaging performance, we developed the “mesoSPIM-ultra”, an upgraded mesoSPIM platform with a temperature-controlled sample chamber, a large field-of-view (FoV) camera and specialized optics to achieve long-working-distance, high-resolution imaging of cleared samples. We applied this approach to investigate the projectome of Chx10 ⁺ neurons, a cell population with complex axonal morphologies along the entire mouse spinal-cord and brain, and implicated in ipsilateral orienting behaviors. By combining behavioral analysis with post-hoc single-neuron reconstructions, we revealed previously inaccessible branching architectures and long-range projections extending from the brainstem to the spinal cord. Together, our work establishes a mechanistic foundation for tissue-clearing and scalable imaging.

Neurological disorders often originate from progressive brain network dysfunctions that start years before symptoms appear. How these changes emerge in the developing human brain remains elusive due to a lack of tractable model systems. Here, we show a cerebral organoid model of Tuberous Sclerosis Complex (TSC) that recapitulates hallmarks of epileptogenesis in vitro. We compare extracellular recordings of TSC organoids with intraoperative electrocorticography from TSC patients to reveal striking functional similarities, including high-frequency oscillations - an electrical biomarker for epileptogenic tissue. In TSC, a human-specific interneuron sub-type derived from the caudal ganglionic eminence drives network hyper-synchronization through increased spontaneous firing and altered excitability. Inhibiting overproliferation of its progenitors via long-term epidermal growth factor receptor inhibition prevented the onset of this pathological phenotype at functional and morphological levels. Our work shows that organoids allow mechanistic analysis of emerging neural network phenotypes, enabling anti-epileptogenic drug testing in a human brain development model.

Background: Single-cell RNA-seq suffers from unwanted technical variation between cells, caused by its complex experiments and shallow sequencing depths. Many conventional normalization methods try to remove this variation by calculating the relative gene expression per cell. However, their choice of the maximum likelihood estimator is not ideal for this application. Results: We present GTestimate, a new normalization method based on the Good–Turing estimator, which improves upon conventional normalization methods by accounting for unobserved genes. To validate GTestimate, we developed a novel cell-targeted PCR amplification approach (cta-seq), which enables ultra-deep sequencing of single cells. Based on these data, we show that the Good–Turing estimator improves relative gene expression estimation and cell–cell distance estimation. Finally, we use GTestimate’s compatibility with Seurat workflows to explore 4 example datasets and show how it can improve downstream results. Conclusion: By choosing a more suitable estimator for the relative gene expression per cell, we were able to improve scRNA-seq normalization, with potentially large implications for downstream results. GTestimate is available as an easy-to-use R-package and compatible with a variety of workflows, which should enable widespread adoption.

As the field of neural organoids and assembloids rapidly expands, there is an emergent need for guidance and advice on designing, conducting and reporting experiments to increase the reproducibility and utility of these models. Here, our consortium– representing specialized laboratories from around the world– presents a framework for the experimental process that ranges from ensuring the quality and integrity of human pluripotent stem cells to characterizing and manipulating neural cells in vitro, and from transplantation techniques to considerations for modeling human development, evolution, and disease. As with all scientific endeavors, we advocate for rigorous experimental designs tailored to explicit scientific questions, and transparent methodologies and data sharing, to provide useful knowledge for both current research practices and for developing regulatory standards.