Development of 3-D Multi-Electrode Arrays for Use with Acute Tissue Slices (original) (raw)
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Complementing single electrode recordings, passive, substrate-integrated thin-film microelectrode arrays (MEAs) have become established tools to investigate spatio-temporal patterns of electrical activity and neuronal interaction in-vitro. In the neurosciences, acute brain slices with accessible and well-preserved neuronal microcircuitry have become the most widespread preparation, that can also be recorded with MEAs for spike activity and local field potentials. Oxygen and nutrients are, however, supplied to the slice tissue by diffusion only (usually from one side) and can may become limiting for tissue stability viability, and slice thickness. Since MEAs record on the face of the slice not directly exposed to the continuous stream of buffer, this might become critical. We therefore developed and compared solid and perforated MEAs for extracellular recording and stimulation, the latter to provide a second exchange surface. For each array we determined the depth profile of the local O 2 -partial pressure (pO 2 ) in cerebellar brain slices. On impermeable MEAs, pO 2 decreased linearly with depth in the tissue. Added diffusion through the perforated MEA surface decreased the slope of the pO 2 gradient and the minimum level reached within the tissue. In addition, signal-to-noise ratios (SNR) in the recordings increased. Improved supply also allows thicker slices and thus the preservation of larger networks, i.e. more complex and in-vivo-like networks. Furthermore, drug accessibility to the recorded cells is improved, accelerating dose-response studies.
Two-dimensional monitoring of spiking networks in acute brain slices
Experimental Brain Research, 2002
To understand spatiotemporally coordinated activity in neural networks and interaction between different areas or layers in brain tissue, simultaneous multisite recording is a prerequisite. For in vitro studies pursuing these goals, substrate integrated, planar microelectrode arrays (MEAs) have been developed to monitor spikes and local field potentials. Here we report for the first time recordings of single-unit spike activity with MEAs in acute slice preparations of the rat cerebellum. We compare these recordings to results of conventional techniques, and discuss the recording conditions in view of the equivalent circuits commonly used. Simultaneous recordings with tungsten microelectrodes and MEAs verified that recording characteristics and signal-to-noise ratios of MEA electrodes were comparable to those of conventional extracellular electrodes. Spike shapes were identical on both electrodes. We found no detectable overlap between spike signals recorded at neighboring MEA electrodes (200 µm spacing). Neuronal spike activity was detected with MEA electrodes at distances of up to 100 µm from the site of spike generation. We conclude that extracellular recording of independent single-unit spike activity with MEAs is indeed suitable to monitor network activity in acute slices, making MEAs an exceptionally useful tool for the assessment of fast network dynamics in acute slices.
Journal of Neuroscience Methods, 1995
Electrical stimulation of nervous tissue with single stimulating electrodes is a technique widely used for the investigation of nervous system function. While it has proved to be useful in all kinds of experiments, single electrode stimuli are: however, far from being 'natural'. In most parts of the living brain, incoming activity results from the firing of a large number of presynaptic neurons, thus reflecting a complex combination of space and time aspects of neural activity. In this paper, a multi-electrode stimulating system is introduced which allows for the generation of fast space-time stimulus patterns. An example for the application of dynamic input patterns to the cerebellar cortex in vitro is given. The corresponding experiments revealed aspects of cerebeliar function which cannot be seen using static or single electrode stimulation.
Microelectrode arrays for stimulation of neural slice preparations
Journal of Neuroscience Methods, 1997
A planar 6×6 array of iridium electrodes with four reference electrodes has been developed for use with neural tissue preparations. Precise knowledge of the relative locations of the array elements allows for spatial neurophysiological analyses. The 10 μm diameter platinized iridium electrodes on a 100 μm pitch have been used to stimulate acutely prepared slices of spinal cord from free-ranging rodents. An intracellular recording from a single neuron in the substantia gelatinosa (SG) using the whole-cell, tight-seal technique allowed low noise, high resolution studies of excitatory or inhibitory electrical responses of a given neuron to inputs from the primary afferent fibers or from stimulation by individual electrodes of the array. The resulting maps of responses provide an indication of the interconnectivity of neural processes. The pattern emerging is that of limited interconnectivity in the SG from areas surrounding a recorded neuron but with strong excitatory or inhibitory effects from those oriented in a longitudinal (rostral–caudal) direction relative to the neuron. The observations to date suggest the neurons of the SG are arranged in sets of independent networks, possibly related to sensory modality and input from particular body regions.
The Journal of Comparative Neurology, 2003
Hippocampal slices often have more synapses than perfusion-fixed hippocampus, but the cause of this synaptogenesis is unclear. Ultrastructural evidence for synaptogenic triggers during slice preparation was investigated in 21-day-old rats. Slices chopped under warm or chilled conditions and fixed after 0, 5, 25, 60, or 180 minutes of incubation in an interface chamber were compared with hippocampi fixed by perfusion or by immersion of the whole hippocampus. There was no significant synaptogenesis in these slices compared with perfusion-fixed hippocampus, but there were other structural changes during slice preparation and recovery in vitro. Whole hippocampus and slices prepared under warm conditions exhibited an increase in axonal coated vesicles, suggesting widespread neurotransmitter release. Glycogen granules were depleted from astrocytes and neurons in 0-min slices, began to reappear by 1 hour, and had fully recovered by 3 hours. Dendritic microtubules were initially disassembled in slices, but reassembled into normal axial arrays after 5 minutes. Microtubules were short at 5 minutes (12.3 Ϯ 1.1 m) but had recovered normal lengths by 3 hours (84.6 Ϯ 20.0 m) compared with perfusion-fixed hippocampus (91 Ϯ 22 m). Microtubules appeared transiently in 15 Ϯ 3% and 9 Ϯ 4% of dendritic spines 5 and 25 minutes after incubation, respectively. Spine microtubules were absent from perfusion-fixed hippocampus and 3-hour slices. Ice-cold dissection and vibratomy in media that blocked activity initially produced less glycogen loss, coated vesicles, and microtubule disassembly. Submersing these slices in normal oxygenated media at 34°C led to glycogen depletion, as well as increased coated vesicles and microtubule disassembly within 1 minute. J. Comp. Neurol. 465:90 -103, 2003.
Preparation of Slice Cultures from Rodent Hippocampus
Cold Spring Harbor protocols, 2017
This protocol describes the preparation of hippocampal slice cultures from rat or mouse pups using sterile conditions that do not require the use of antibiotics or antimycotics. Combining very good optical and electrophysiological accessibility with a lifetime approaching that of the intact animal, many fundamental questions about synaptic plasticity and long-term dynamics of network connectivity can be addressed with this preparation.
Journal of neuroscience methods, 2012
We designed an electrophysiology system to record from 16 separate brain slices simultaneously. The system is able to obtain stable, extracellular responses from the CA1 region of the hippocampus. The system is able to record NMDA-dependent long-term potentiation. The system can record functional loss and recovery during an oxygen and glucose deprivation insult. The system is both cost-and space-efficient, reduces data variability and minimizes animal use.
Pfl�gers Archiv European Journal of Physiology, 1989
(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.