Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

AbstractThe salamander is a key limbed vertebrate from which many major scientific questions can be addressed in the fields of motor control, evolutionary biology, and regeneration biology. An important gap of knowledge is the description of the electrophysiological properties of the neurons constituting their central nervous system. To our knowledge, some patch-clamp electrophysiological recordings were done in the spinal cord and recently in hindbrain slices, but not in any higher brain region. Here, we present a method to obtain patch-clamp recordings in slices of the telencephalon, diencephalon and rhombencephalon of salamanders. The method includes dissection of the brain, brain slice preparation, visual identification of neurons and patch-clamp recordings. We provide single cell recordings in the rhombencephalon, diencephalon and telencephalon of salamanders. This method should open new avenues to dissect the operation of salamander brain circuits at the cellular level.Highlights- Salamander brain slices of telencephalon, diencephalon, and rhombencephalon- Patch-clamp recordings in salamander brain slices- The salamander as a model to decipher tetrapod neural microcircuits

Download Full-text

Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization

eLife ◽

10.7554/elife.65482 ◽

2021 ◽

Vol 10 ◽

Author(s):

Brian R Lee ◽

Agata Budzillo ◽

Kristen Hadley ◽

Jeremy A Miller ◽

Tim Jarsky ◽

...

Keyword(s):

Patch Clamp ◽

Mammalian Species ◽

Membrane Integrity ◽

Brain Slices ◽

Brain Regions ◽

Quality Data ◽

Key Factors ◽

Patch Clamp Technique ◽

Online Quality Control ◽

Patch Clamp Electrophysiology

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.

Download Full-text