High-throughput combined voltage-clamp/current-clamp analysis of freshly isolated neurons

Functionally-selective inhibition of threshold sodium currents and excitability in dorsal root ganglion neurons by cannabinol

“…9d–i ). As noted, each of these three midpoints are indicative of different amounts and combinations of Nav channels; therefore, the lack of statistical significance at the most depolarized midpoint (V D2 ), which is likely dominated by Nav1.8 32 , after shortest duration of 500 ms, suggests that CBN functionally targets and stabilizes Nav inactivation, which inhibits Nav currents. This further suggests that it does not have structural selectivity among Nav channels.…”

“…Specifically, DRG neurons across various diameters express different stoichiometric combinations of Nav channels, with characteristic biophysical signatures (e.g., kinetic and voltage-dependent properties). We recently developed a new robotic automated high-throughput method to investigate the biophysical properties of diverse neurons, compared head-to-head and simultaneously, immediately after dissociation and isolation of cells from intact animal tissue 32 . We used this method to investigate the effects of 15 µM CBN on primary DRG neurons across various sizes obtained from rat pups.…”

“…MEA recordings in intact neurons measure activity recorded extracellularly from larger numbers of neurons, but do not provide an assessment of action potentials. Constraints on the throughput of manual patch-clamp have been overcome using automated patch-clamping 20 , 21 , 32 , 52 . However, as usually performed, automated patch-clamp analysis is generally used to assess ion channels or receptors expressed in cell lines, and is not practicable for native neurons; this is particularly important since the properties of ion channels can be different following expression in cell lines versus expression within neurons 64 .…”

“…In the present study, we assessed the effects of CBN on Nav1.7 and on DRG neuron excitability using these three approaches. In addition to the automated patch-clamp analysis of Nav1.7 channels expressed in a cell line and MEA analysis of DRG neurons, we used a newly developed method for automated high-throughput patch-clamp analysis that permits study of freshly isolated DRG neurons 32 . In addition to providing a basis for high-throughput analysis of multiple cells in a simultaneous comparative manner, this new methodology permits assessment of these cells by both voltage-clamp (Figs.…”

Compartment-specific regulation of NaV1.7 in sensory neurons after acute exposure to TNF-α

“…Step-by-step protocols for the operation of these high through-put machines is described elsewhere. 84 …”