Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization
- Allen Institute, United States
- Byte Physics e. K., Germany
Abstract
The Patch-seq approach is a powerful variation of the patch-clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at scale, we identified and refined key factors that contribute to the efficient collection of high-quality data. We developed patch-clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized the importance of extracting the nucleus for transcriptomic success and maximizing membrane integrity during nucleus extraction for morphology success. The protocol is generalizable to different species and brain regions, as demonstrated by capturing multimodal data from human and macaque brain slices. The protocol, analysis and acquisition software are compiled at https://github.com/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate data across diverse mammalian species and that is compatible with large publicly available Patch-seq datasets.
Data availability
The data used in this manuscript, the software packages, the detailed protocol, and online resources are freely available to the public and have been consolidated at https://github.com/AllenInstitute/patchseqtools.
Article and author information
Author details
Funding
NIH Office of the Director (P51OD010425)
- Brian E Kalmbach
- Jonathan T Ting
National Center for Advancing Translational Sciences (UL1TR000423)
- Brian E Kalmbach
- Jonathan T Ting
National Institute of Mental Health (U01 MH114812-02)
- Ed Lein
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: The animal research in this study was performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Public Health Service Policy on Humane Care and Use of Laboratory Animals in compliance with National Institutes of Health policy. All housing, handling, and experimental use of the animals occurred with the oversight and approval of the Allen Institute Institutional Animal Care and Use Committee (Protocol 1809). All surgeries and retro-orbital injections were performed under isoflurane anesthesia with perioperative analgesics and fluid support.
Human subjects: De-identified human brain tissue and data used in this research was collected by local hospitals during clinically necessary surgery. Study participants gave informed consent to share their de-identified tissue and data either with the Allen Institute specifically or more broadly with collaborators of the study PIs prior to surgery. Participants consented to share their de-identified genomic data in controlled access in compliance with National Institutes of Health Genomic Data Sharing policy. The study participants were informed that the resulting data might be broadly shared, through publications, presentations, or scientific repositories and of the potential risks of sharing these data. Samples obtained from the Swedish Neuroscience Institute were collected under approved Western Institutional Review Board protocols (#1111798 and #1068035) in collaboration with Drs. Charles Cobb and Ryder Gwinn respectively. Samples obtained from Harborview Medical Center were obtained under approval of the University of Washington Institutional Review Board protocol (#HSD No. 49119) in collaboration with Dr. Jeffrey Ojemann.
Copyright
© 2021, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 44
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization
eLife 10:e65482.
https://doi.org/10.7554/eLife.65482
Further reading
-
- Genetics and Genomics
Resistance to anthelmintics, particularly the macrocyclic lactone ivermectin (IVM), presents a substantial global challenge for parasite control. We found that the functional loss of an evolutionarily conserved E3 ubiquitin ligase, UBR-1, leads to IVM resistance in Caenorhabditis elegans. Multiple IVM-inhibiting activities, including viability, body size, pharyngeal pumping, and locomotion, were significantly ameliorated in various ubr-1 mutants. Interestingly, exogenous application of glutamate induces IVM resistance in wild-type animals. The sensitivity of all IVM-affected phenotypes of ubr-1 is restored by eliminating proteins associated with glutamate metabolism or signaling: GOT-1, a transaminase that converts aspartate to glutamate, and EAT-4, a vesicular glutamate transporter. We demonstrated that IVM-targeted GluCls (glutamate-gated chloride channels) are downregulated and that the IVM-mediated inhibition of serotonin-activated pharynx Ca2+ activity is diminished in ubr-1. Additionally, enhancing glutamate uptake in ubr-1 mutants through ceftriaxone completely restored their IVM sensitivity. Therefore, UBR-1 deficiency-mediated aberrant glutamate signaling leads to ivermectin resistance in C. elegans.
-
- Genetics and Genomics
Osteoporosis, characterized by reduced bone density and strength, increases fracture risk, pain, and limits mobility. Established therapies of parathyroid hormone (PTH) analogs effectively promote bone formation and reduce fractures in severe osteoporosis, but their use is limited by potential adverse effects. In the pursuit of safer osteoporosis treatments, we investigated R25CPTH, a PTH variant wherein the native arginine at position 25 is substituted by cysteine. These studies were prompted by our finding of high bone mineral density in a hypoparathyroidism patient with the R25C homozygous mutation, and we explored its effects on PTH type-1 receptor (PTH1R) signaling in cells and bone metabolism in mice. Our findings indicate that R25CPTH(1–84) forms dimers both intracellularly and extracellularly, and the synthetic dimeric peptide, R25CPTH(1–34), exhibits altered activity in PTH1R-mediated cyclic AMP (cAMP) response. Upon a single injection in mice, dimeric R25CPTH(1–34) induced acute calcemic and phosphaturic responses comparable to PTH(1–34). Furthermore, repeated daily injections increased calvarial bone thickness in intact mice and improved trabecular and cortical bone parameters in ovariectomized (OVX) mice, akin to PTH(1–34). The overall results reveal a capacity of a dimeric PTH peptide ligand to activate the PTH1R in vitro and in vivo as PTH, suggesting a potential path of therapeutic PTH analog development.
