A robot for high yield electrophysiology and morphology of single neurons in vivo

Abstract

Single-cell characterization and perturbation of neurons provides knowledge critical to addressing fundamental neuroscience questions including the structure–function relationship and neuronal cell-type classification. Here we report a robot for efficiently performing in vivo single-cell experiments in deep brain tissues optically difficult to access. This robot automates blind (non-visually guided) single-cell electroporation (SCE) and extracellular electrophysiology, and can be used to characterize neuronal morphological and physiological properties of, and/or manipulate genetic/chemical contents via delivering extraneous materials (for example, genes) into single neurons in vivo. Tested in the mouse brain, our robot successfully reveals the full morphology of single-infragranular neurons recorded in multiple neocortical regions, as well as deep brain structures such as hippocampal CA3, with high efficiency. Our robot thus can greatly facilitate the study of in vivo full morphology and electrophysiology of single neurons in the brain.

Brain-wide single neuron reconstruction reveals morphological diversity in molecularly defined striatal, thalamic, cortical and claustral neuron types

“…Full reconstruction of a single neuron requires sparse, robust and consistent neuronal labeling. Previous genetic approaches applied to produce sparse or single neuron labeling, including viral delivery (e.g., using sindbis virus or AAV) and in vivo electroporation [22][23][24][26][27][28][29][30][31]37 , often resulted in substantial cell-to-cell and animal-to-animal variations, and were usually restricted to few brain regions. In rare cases, extremely sparse labeling was obtained utilizing the low recombination efficiency of CreER driver lines and a sensitive, alkaline phosphatase-based histochemical reporter 25,41 .…”

“…Despite its importance, data on single neuron axonal morphologies are currently lacking for most projection neuron types in mammals, in large part because axons often cover large distances and are severed in ex vivo brain slices. Previous efforts have been made in rodents to fully label single neurons with small molecules or fluorescent proteins through in vivo whole-cell patching, in vivo electroporation [22][23][24] , sparse transgenic labeling 25 , or sparse viral labeling with sindbis virus [26][27][28] or adeno-associated virus (AAV) [29][30][31] . Conventionally this is followed by serial sectioning, imaging of each section, and manual reconstruction of the labeled neurons across many consecutive sections.…”

Single‐Cell Mass Spectrometry Approaches to Explore Cellular Heterogeneity

“…Live-cell sampling in LAESI occurs on the millisecond timescale under ambient conditions,t hereby maintaining the cells in their native state until right before analysis. [130][131] This approach can be combined with capillary microsampling followed by ESI-MS analysis in ar apid and controlled fashion. [129] In situ sampling with probes,such as capillary microsampling,enables direct extraction of cell contents.…”

Einzelzell‐Massenspektrometrie zur Untersuchung zellulärer Heterogenität

“…Zur Steigerung von experimentellem Durchsatz und Reproduzierbarkeit können das robotergesteuerte kapillare Mikrosampling und die automatisierte Laserablation untersucht werden. Beispielsweise wurden mit der robotergesteuerten Patch‐Clamp‐Technik spezifische Einzelneuronen anvisiert und ihre elektrophysiologischen Bedingungen gemessen . Diese Methode kann mit dem kapillaren Mikrosampling und anschließender ESI‐MS‐Analyse schnell und kontrolliert kombiniert werden.…”