Patch-clamp recording in brain slices with improved slicer technology

Abstract

The use of advanced patch-clamp recording techniques in brain slices, such as simultaneous recording from multiple neurons and recording from dendrites or presynaptic terminals, demands slices of the highest quality. In this context the mechanics of the tissue slicer are an important factor. Ideally, a tissue slicer should generate large-amplitude and high-frequency movements of the cutting blade in a horizontal axis, with minimal vibrations in the vertical axis. We developed a vibroslicer that fulfils these in part conflicting requirements. The oscillator is a permanent-magnet-coil-leaf-spring system. Using an auto-resonant mechano-electrical feedback circuit, large horizontal oscillations (up to 3 mm peak-to-peak) with high frequency ( approximately 90 Hz) are generated. To minimize vertical vibrations, an adjustment mechanism was employed that allowed alignment of the cutting edge of the blade with the major axis of the oscillation. A vibroprobe device was used to monitor vertical vibrations during adjustment. The system is based on the shading of the light path between a light-emitting diode (LED) and a photodiode. Vibroprobe monitoring revealed that the vibroslicer, after appropriate adjustment, generated vertical vibrations of <1 microm, significantly less than many commercial tissue slicers. Light- and electron-microscopic analysis of surface layers of slices cut with the vibroslicer showed that cellular elements, dendritic processes and presynaptic terminals are well preserved under these conditions, as required for patch-clamp recording from these structures.

A large pool of releasable vesicles in a cortical glutamatergic synapse

“…No correction was made for shrinkage. The diameter of the axon was 0.14-0.40 m, consistent with electron-microscopic data (34). After conversion into NEURON format, access resistance, shunt conductance, and specific parameters C m , R i , and R m were determined by direct fitting of the current transients (37) with Fitpraxis of NEURON 5.1 (ref.…”

“…Transverse 250-to 300-m-thick slices were cut from the hippocampi of 20-to 26-day-old Wistar rats with a custom-built vibratome (34). The animals were killed by decapitation in accordance with national and institutional guidelines.…”

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

“…Presynaptic action potential and voltage-gated Na + and K + channels in hippocampal mossy fiber boutons After the development of methods for patch-clamp recording in brain slices [26,38], techniques were gradually refined to a point when it became possible to obtain recordings from presynaptic elements, including mossy fiber boutons [12,28,34]. The key steps of improvement were as follows: (1) the enhanced visibility provided by high-numerical aperture objectives and infrared illumination [75]; (2) the development of vibratomes with minimal blade vibration in the Fig.…”

“…Arrows indicate mossy fiber axons, arrowheads show filopodial extensions, which contact interneuron dendrites. Illustration from Acsády et al [1], with permission of the Society for Neuroscience, copyright (1998) vertical direction, resulting in an improved preservation of superficial tissue layers [34], although mossy fiber terminal recordings with commercially available tissue slicers were also reported [72]; (3) the use of sucrose-based cutting solutions with reduced Na + and Ca 2+ concentrations, which further improves tissue preservation during cutting and maintenance of slices [34]. Collectively, these refinements made it possible to routinely obtain patch-clamp recordings from large hippocampal mossy fiber boutons in the stratum lucidum [6,27,33,37].…”

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

“…We find that the use of sucrose-ACSF, whereby the overall sodium concentration and thus excitability of neurons is reduced, dramatically improves the quality of the slices and interneuron survival 18 . However, it should be highlighted that many researchers have made use of standard recording ACSF for slice preparation, to great effect 10,11,15 .…”