The touch and zap method for in vivo whole-cell patch recording of intrinsic and visual responses of cortical neurons and Glial cells

Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe "Touch and Zap", an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the "Touch". By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or "Zap", as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi-automatically, this approach is more reproducible and less dependent on experimenter technique

Dynaflow ™ 48, a microfluidic chip solution for increasing throughput and data quality in patch-clamp-based drug screening

Ion channels are transm embrane proteins, found in virtually all cell types throughout the human body. Ion channels underlie neural communication, memory, behavior, every movement and heartbeat, and are as such prone to cause disease if malfunctioning. Therefore ion channels are very important targets in drug discovery. The gold standard technique for obtaining information on ion channel function with high information content and temporal resolution is patch-clamp. The technique measures the minute currents originating from the movement of ions across the cellular membrane, and enables determination of the potency and efficacy of a drug. However, patch-clamp suffers from serious throughput restrictions due to its laborious nature. To address the throughput problems we have developed a microfluidic chip containing 48 microchannels for an extremely rapid, sequential delivery of a large number of completely controlled solution environments to a lifted, patch-clamped cell. In this way, throughput is increased drastically compared to classical patch-clamp perfusion set-ups, with uncompromised data quality. The 48-microchannel chip has been used for the characterization of drugs affecting ligand-gated ion channels including agonists, antagonists and positive modulators with positive effects on both throughput and data quality.Zadanie pt. „Digitalizacja i udostępnienie w Cyfrowym Repozytorium Uniwersytetu Łódzkiego kolekcji czasopism naukowych wydawanych przez Uniwersytet Łódzki” nr 885/P-DUN/2014 dofinansowane zostało ze środków MNiSW w ramach działalności upowszechniającej naukę

Batch Fabrication of High-Performance Planar Patch-Clamp Devices in Quartz

The success of the patch-clamp technique has driven an effort to create wafer-based patch-clamp platforms. We develop a lithographic/electrochemical processing scheme that generates ultrasmooth, high aspect ratio pores in quartz. These devices achieve gigaohm seals in nearly 80% of trials, with the majority exhibiting seal resistances from 20-80 GΩ, competing with pipette-based patch-clamp measurements

Electrophysiological analysis of mammalian cells expressing hERG using automated 384-well-patch-clamp

BACKGROUND: An in vitro electrophysiological assay system, which can assess compound effects and thus show cardiotoxicity including arrhythmia risks of test drugs, is an essential method in the field of drug development and toxicology. METHODS: In this study, high-throughput electrophysiological recordings of human embryonic kidney (HEK 293) cells and Chinese hamster ovary (CHO) cells stably expressing human ether-a-go-go related gene (hERG) were performed utilizing an automated 384-well-patch-clamp system, which records up to 384 cells simultaneously. hERG channel inhibition, which is closely related to a drug-induced QT prolongation and is increasing the risk of sudden cardiac death, was investigated in the high-throughput screening patch-clamp system. RESULTS: In the automated patch-clamp measurements performed here, K(v) currents were investigated with high efficiency. Various hERG channel blockers showed concentration-dependent inhibition, the 50 % inhibitory concentrations (IC(50)) of those blockers were in good agreement with previous reports. CONCLUSIONS: The high-throughput patch-clamp system has a high potential in the field of pharmacology, toxicology, and cardiac physiology, and will contribute to the acceleration of pharmaceutical drug development and drug safety testing

Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels

Important modulatory roles have been attributed to presynaptic NMDA receptors (NMDARs) located on cerebellar interneuron terminals. Evidence supporting a presynaptic location includes an increase in the frequency of mini events following the application of NMDA and gold particle-labelled NMDA receptor antibody localisation. However, more recent work, using calcium indicators, casts doubt on the idea of presynaptic NMDARs because basket cell varicosities did not show the expected calcium rise following either the local iontophoresis of L-aspartate or the two-photon uncaging of glutamate. (In theory such calcium imaging is sensitive enough to detect the calcium rise from even a single activated receptor.) It has therefore been suggested that the effects of NMDA are mediated via the activation of somatodendritic channels, which subsequently cause a subthreshold depolarization of the axon. Here we report results from a vibrodissociated preparation of cerebellar Purkinje cells, in which the interneuron cell bodies are no longer connected but many of their terminal varicosities remain attached and functional. This preparation can retain both inhibitory and excitatory inputs. We find that the application of NMDA increases the frequency of both types of synaptic event. The characteristics of these events suggest they can originate from interneuron, parallel fiber and even climbing fiber terminals. Interestingly, retrograde signalling seems to activate only the inhibitory terminals. Finally, antibody staining of these cells shows NMDAR-like immunoreactivity co-localised with synaptic markers. Since the Purkinje cells show no evidence of postsynaptic NMDAR-mediated currents, we conclude that functional NMDA receptors are located on presynaptic terminals