Published: Hippocampal transcriptomic responses to enzyme‐mediated cellular dissociation

The manuscript associated with this GitHub repository was published in Hippocampus. It is available online at https://onlinelibrary.wiley.com/doi/10.1002/hipo.23095

GitHub repository for hippocampal transcriptomic responses to cellular dissociation

Single-neuron gene expression studies may be especially important for understanding nervous system structure and function because of the neuron-specific functionality and plasticity that defines functional neural circuits. Cellular dissociation is a prerequisite technical manipulation for single-cell and single cell-population studies, but the extent to which the cellular dissociation process affects neural gene expression has not been determined. This information is necessary for interpreting the results of experimental manipulations that affect neural function such as learning and memory. The goal of this research was to determine the impact of chemical cell dissociation on brain transcriptomes. We compared gene expression of microdissected samples from the dentate gyrus (DG), CA3, and CA1 subfields of the mouse hippocampus either prepared by a standard tissue homogenization protocol or subjected to a chemical cellular dissociation procedure. We report that compared to homogenization, chemical cellular dissociation alters about 350 genes or 2% of the hippocampal transcriptome. Few genes canonically implicated in long-term potentiation (LTP) and fear memory change expression levels in response to the dissociation procedure. Nevertheless, sample preparation did affect gene expression profiles, which might confound interpretation of results depending on the research question. This study is important for the investigation of any complex tissues as research effort moves from subfield level analysis to single cell analysis of gene expression.

The GitHub repository for analyses of hippocampal transcriptomic responses to technical and biological perturbations

Many factors contribute to variation in gene expression. These data were collected to examine common sources of variation in gene expression that are influenced by the experimental design. How does technique for sample collection affect neural gene expression? How does the stress accompanying cognitive paradigms alter transcription? In answer to these questions, we identified are largely distinct response of the hippocampus transcriptome to specific perturbations, with some important confounding overlap. It is encouraging that the overlap between cellular dissociation and cognitive training is small, indicating that these technical and biological processes affect different transcriptional processes. It is also encouraging to know that the stressful experience had no substantial effect on hippocampal gene expression, which if generalizable to other tasks will allow for using behavioral control groups and behavioral manipulations that also induce modest, potentially confounding stress. Our bioinformatic workflow is reproducible and incorporates public data from a published study that cataloged subfield-specific gene expression in the hippocampus. These findings provide insight into how cellular and biological manipulations influence gene expression.

The GitHub repository for an integrative analysis of genomic plasticity in the hippocampus

Cost-effective next-generation sequencing has made unbiased gene expression investigations possible. Gene expression studies at the level of single neurons may be especially important for understanding nervous system structure and function because of neuron-specific functionality and plasticity. While cellular dissociation is a prerequisite technical manipulation for such single-cell studies, the extent to which the process of dissociating cells affects neural gene expression has not been determined. Here, we examine the effect of cellular dissociation on gene expression in the mouse hippocampus. We also determine to which extent such changes might confound studies on the behavioral and physiological functions of hippocampus.

This dataset contains the data, software, and results the accompany a manuscript that is in the process of submission to the journal Hippocampus.