Search for: All records

SUMMARY Single cell genomics has revolutionized our understanding of neuronal cell types. However, scalable technologies for probing single-cell connectivity are lacking, and we are just beginning to understand how molecularly defined cell types are organized into functional circuits. Here, we describe a protocol to generate high-complexity barcoded rabies virus (RV) for scalable circuit mapping from tens of thousands of individual starter cells in parallel. In addition, we introduce a strategy for targeting RV-encoded barcode transcripts to the nucleus so that they can be read out using single-nucleus RNA sequencing (snRNA-seq). We apply this tool in organotypic slice cultures of the developing human cerebral cortex, which reveals the emergence of cell type– specific circuit motifs in midgestation. By leveraging the power and throughput of single cell genomics for mapping synaptic connectivity, we chart a path forward for scalable circuit mapping of molecularly-defined cell types in healthy and disease states. 

SUMMARY Electrophysiology offers a high-resolution method for real-time measurement of neural activity. Longitudinal recordings from high-density microelectrode arrays (HD-MEAs) can be of considerable size for local storage and of substantial complexity for extracting neural features and network dynamics. Analysis is often demanding due to the need for multiple software tools with different runtime dependencies. To address these challenges, we developed an open-source cloud-based pipeline to store, analyze, and visualize neuronal electrophysiology recordings from HD-MEAs. This pipeline is dependency agnostic by utilizing cloud storage, cloud computing resources, and an Internet of Things messaging protocol. We containerized the services and algorithms to serve as scalable and flexible building blocks within the pipeline. In this paper, we applied this pipeline on two types of cultures, cortical organoids andex vivobrain slice recordings to show that this pipeline simplifies the data analysis process and facilitates understanding neuronal activity. 

Abstract Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods. 

Abstract Objective.Neural activity represents a functional readout of neurons that is increasingly important to monitor in a wide range of experiments. Extracellular recordings have emerged as a powerful technique for measuring neural activity because these methods do not lead to the destruction or degradation of the cells being measured. Current approaches to electrophysiology have a low throughput of experiments due to manual supervision and expensive equipment. This bottleneck limits broader inferences that can be achieved with numerous long-term recorded samples.Approach.We developed Piphys, an inexpensive open source neurophysiological recording platform that consists of both hardware and software. It is easily accessed and controlled via a standard web interface through Internet of Things (IoT) protocols.Main results.We used a Raspberry Pi as the primary processing device along with an Intan bioamplifier. We designed a hardware expansion circuit board and software to enable voltage sampling and user interaction. This standalone system was validated with primary human neurons, showing reliability in collecting neural activity in near real-time.Significance.The hardware modules and cloud software allow for remote control of neural recording experiments as well as horizontal scalability, enabling long-term observations of development, organization, and neural activity at scale. 

INTRODUCTION Balance between excitatory and inhibitory neuron (interneuron) populations in the cortex promotes normal brain function. Interneurons are primarily generated in the medial, caudal, and lateral ganglionic eminences (MGE, CGE, and LGE) of the ventral embryonic forebrain; these subregions give rise to distinct interneuron subpopulations. In rodents, the MGE generates cortical interneurons, the parvalbumin + (PV + ) and somatostatin + (SST + ) subtypes that connect with excitatory neurons to regulate their activity. Defects in interneuron production have been implicated in neurodevelopmental and psychiatric disorders including autism, epilepsy, and schizophrenia. RATIONALE How does the human MGE (hMGE) produce the number of interneurons required to populate the forebrain? The hMGE contains progenitor clusters distinct from what has been observed in the rodent MGE and other germinal zones of the human brain. This cytoarchitecture could be the key to understanding interneuron neurogenesis. We investigated the cellular and molecular properties of different compartments within the developing hMGE, from 14 gestational weeks (GW) to 39 GW (term), to study their contribution to the production of inhibitory interneurons. We developed a xenotransplantation assay to follow the migration and maturation of the human interneurons derived from this germinal region. RESULTS Within the hMGE, densely packed aggregates (nests) of doublecortin + (DCX + ) and LHX6 + cells were surrounded by nestin + progenitor cells and their processes. These DCX + cell–enriched nests (DENs) were observed in the hMGE but not in the adjacent LGE. We found that cells within DENs expressed molecular markers associated with young neurons, such as DCX, and polysialylated neural cell adhesion molecule (PSA-NCAM). A subpopulation also expressed Ki-67, a marker of proliferation; therefore, we refer to these cells as neuroblasts. A fraction of DCX + cells inside DENs expressed SOX2 and E2F1, transcription factors associated with progenitor and proliferative properties. More than 20% of DCX + cells in the hMGE were dividing, specifically within DENs. Proliferating neuroblasts in DENs persisted in the hMGE throughout prenatal human brain development. The division of DCX + cells was confirmed by transmission electron microscopy and time-lapse microscopy. Electron microscopy revealed adhesion contacts between cells within DENs, providing multiple sites to anchor DEN cells together. Neuroblasts within DENs express PCDH19, and nestin + progenitors surrounding DENs express PCDH10; these findings suggest a role for differential cell adhesion in DEN formation and maintenance. When transplanted into the neonatal mouse brain, dissociated hMGE cells reformed DENs containing proliferative DCX + cells, similar to DENs observed in the prenatal human brain. This suggests that DENs are generated by cell-autonomous mechanisms. In addition to forming DENs, transplanted hMGE-derived neuroblasts generated young neurons that migrated extensively into cortical and subcortical regions in the host mouse brain. One year after transplantation, these neuroblasts had differentiated into distinct γ-aminobutyric acid–expressing (GABAergic) interneuron subtypes, including SST + and PV + cells, that showed morphological and functional maturation. CONCLUSION The hMGE harbors DENs, where cells expressing early neuronal markers continue to divide and produce GABAergic interneurons. This MGE-specific arrangement of neuroblasts in the human brain is present until birth, supporting expanded neurogenesis for inhibitory neurons. Given the robust neurogenic output from this region, knowledge of the mechanisms underlying cortical interneuron production in the hMGE will provide insights into the cell types and developmental periods that are most vulnerable to genetic or environmental insults. Nests of DCX + cells in the ventral prenatal brain. Schematic of a coronal view of the embryonic human forebrain showing the medial ganglionic eminence (MGE, green), with nests of DCX + cells (DENs, green). Nestin + progenitor cells (blue) are present within the VZ and iSVZ and are intercalated in the oSVZ (where DENs reside). The initial segment of the oSVZ contains palisades of nestin + progenitors referred to as type I clusters (light blue cells) around DENs. In the outer part of the oSVZ, DENs transition to chains of migrating DCX + cells; surrounding nestin + progenitors are arranged into groups of cells referred to as type II clusters (white cells). In addition to proliferation of nestin + progenitors, cell division is present among DCX + cells within DENs, suggesting multiple progenitor states for the generation of MGE-derived interneurons in the human forebrain. ILLUSTRATION: NOEL SIRIVANSANTI