<i>dotdotdot</i>: an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues

Spangler

et al. 2020

Preprint

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Single cell/nucleus technologies are powerful tools to study cell type-specific expression in the human brain, but most large-scale efforts have focused on characterizing cortical brain regions and their constituent cell types. However, additional brain regions - particularly those embedded in basal ganglia and limbic circuits - play important roles in neuropsychiatric disorders and addiction, suggesting a critical need to better understand their molecular characteristics. We therefore created a single-nucleus RNA-sequencing (snRNA-seq) resource across five human brain regions (hippocampus, HPC; dorsolateral prefrontal cortex, DLPFC; subgenual anterior cingulate cortex, sACC; nucleus accumbens, NAc; and amygdala, AMY), with emphasis on the NAc and AMY, given their involvement in reward signaling and emotional processing. We identified distinct and potentially novel neuronal subpopulations, which we validated by smFISH for various subclasses of NAc interneurons and medium spiny neurons (MSNs). We additionally benchmarked these datasets against published datasets for corresponding regions in rodent models to define cross-species convergence and divergence across analogous cell subclasses. We characterized the transcriptomic architecture of regionally-defined neuronal subpopulations, which revealed strong patterns of similarities in specific neuronal subclasses across the five profiled regions. Finally, we measured genetic associations between risk for psychiatric disease and substance use behaviors with each of the regionally-defined cell types. This analysis further supported NAc and AMY involvement in risk for psychiatric illness by implicating specific neuronal subpopulations, and highlighted potential involvement of an MSN population associated with stress signaling in genetic risk for substance use.

Section: Identification Of Refined Medium Spiny Neuron Subpopulationsmentioning

confidence: 96%

Section: Rnascope Single Molecule Fluorescent In Situ Hybridization (mentioning

confidence: 99%

Single-nucleus transcriptome analysis reveals cell type-specific molecular signatures across reward circuitry in the human brain

Tran

Spangler

et al. 2020

Preprint

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“…For each subject, a cortical strip was tile imaged at 20x to capture L1 to WM. Following image acquisition, lambda stacks in z -series were linearly unmixed in Zen software (weighted; no autoscale) using reference emission spectral profiles previously created in Zen (Maynard et al, 2019) , stitched, maximum intensity projected, and saved as Carl Zeiss Image " .czi " files.…”

Section: Rnascope Single Molecule Fluorescent In Situ Hybridization (mentioning

confidence: 99%

“…Because it is considered a gold standard for quantifying gene expression with high spatial resolution, we recently established and optimized methods for using multiplex single-molecule fluorescent in situ hybridization (smFISH) in postmortem human brain tissue (Maynard et al, 2019) . However, multiplexing with these technologies is limited, and even for methodologies that can accommodate hundreds to thousands of transcripts simultaneously, molecular crowding within cells leads to fluorescence overlap, which introduces significant microscopy-related issues and computational challenges (Burgess, 2019;Lein et al, 2017) .…”

Section: Introductionmentioning

confidence: 99%

Transcriptome-scale spatial gene expression in the human dorsolateral prefrontal cortex

Collado‐Torres

Weber³

et al. 2020

Preprint

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140

326

We used the 10x Genomics Visium platform to define the spatial topography of gene expression in the six-layered human dorsolateral prefrontal cortex (DLPFC). We identified extensive layer-enriched expression signatures, and refined associations to previous laminar markers. We overlaid our laminar expression signatures onto large-scale single nuclei RNA sequencing data, enhancing spatial annotation of expression-driven clusters. By integrating neuropsychiatric disorder gene sets, we showed differential layer-enriched expression of genes associated with schizophrenia and autism spectrum disorder, highlighting the clinical relevance of spatially-defined expression. We then developed a data-driven framework to define unsupervised clusters in spatial transcriptomics data, which can be applied to other tissues or brain regions where morphological architecture is not as well-defined as cortical laminae. We lastly created a web application for the scientific community to explore these raw and summarized data to augment ongoing neuroscience and spatial transcriptomics research ( http://research.libd.org/spatialLIBD ).

“…Once centers and boundaries of individual cells were isolated, an intensity threshold was set for transcript detection, and watershed analysis was used to split the remaining pixels in each channel into identified transcripts. Custom MATLAB functions 49 were then used to determine the size of each detected transcript (regionprops3 function in Image Processing toolbox), and split unusually large areas of fluorescence into multiple transcripts based on known transcript size. Each transcript was then assigned to a cell based on its position in 3 dimensions; transcripts that were detected outside of the boundaries of a cell were excluded from further analysis.…”

Section: Single-molecule In Situ Hybridizationmentioning

confidence: 99%

Molecularly-Defined Hippocampal Inputs Regulate Population Dynamics in the Prelimbic Cortex to Suppress Context Fear Memory Recall

Hallock

Quillian

et al. 2019

Preprint

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Associating fearful events with the context in which they occur is critical for survival. Dysregulation of contextfear memory processing is a hallmark symptom of several neuropsychiatric disorders, including generalized anxiety disorder (GAD) and post-traumatic stress disorder (PTSD). Both the hippocampus and prelimbic subregion (PrL) of the medial prefrontal cortex (mPFC) have been linked with context fear memory recall in rodents, but the mechanisms by which hippocampal-prelimbic circuitry regulates this process remains poorly understood. Spatial and genetic targeting of this circuit in mice allowed us to use molecular profiling to show that hippocampal neurons with projections to the PrL (vHC-PrL projectors) are a transcriptomically-distinct subpopulation that is enriched for expression of genes associated with both GAD and PTSD. We further show that stimulation of this population of vHC-PrL projectors suppresses context fear memory recall and impairs the ability of PrL neurons to dynamically distinguish between distinct phases of fear learning. Using transgenic and circuit-specific molecular targeting approaches, we demonstrate that unique patterns of activity-dependent gene transcription within vHC-PrL projectors causally regulate excitatory/inhibitory balance in the PrL during context fear memory recall. Together, our data illuminate the molecular mechanisms by which hippocampalprelimbic circuitry regulates the retrieval of contextually-mediated fear memories.