The brain slice preparation: a tribute to the pioneer Henry McIlwain

doi:10.1016/0165-0270(94)00187-L

Journal of Neuroscience Methods

Volume 59, Issue 1, June 1995, Pages 5-9

https://doi.org/10.1016/0165-0270(94)00187-L Get rights and content

Abstract

Henry McIlwain is usually remembered for his major contributions to the discipline of neurochemistry. He also had a profound infuence on electrophysiology by inventing and establishing the brain slice technique. This article describes some of his pioneering studies where he devised ways of preparing, both manually and mechanically (using the McIlwain tissue chopper), viable slices of mammalian cortical tissue and showed how these could be maintained in a brain slice chamber. His work with Choh-Luh Li (Li and McIlwain, 1957) on the first intracellular recording from brain slices and his work with Chosaburo Yamamoto (Yamamoto and McIlwain, 1966) describing the first study of synaptic transmission in brain slices are highlighted. In these studies, McIlwain started the use of brain slices to investigate functional anatomy, to study the effects of hypoxia and hypoglycaemia, to perform quantitative pharmacological analyses and to study synaptic plasticity: investigations which continue today to provide important insights into the functioning of the brain in health and disease.

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For example, administration of isoflurane anesthesia prior to decapitation (Pekny et al., 2014), or changes in brain circuitry induced by brain slice preparation itself (Ting et al., 2014), might have influenced susceptibility to block by CPP. While the many possible changes are impossible to discount, we would note that brain slice physiology has been used widely for decades in investigations of cellular and network properties that underlie brain function (Collingridge, 1995), and it has been found to be generally reliable. A final type of technical artefact to consider is whether the concentration of CPP required to block NMDARs in vitro could have been influenced by the method we used to measure NMDAR-fEPSPs
Drugs that block N-methyl-d-aspartate receptors (NMDARs) suppress hippocampus-dependent memory formation; they also block long-term potentiation (LTP), a cellular model of learning and memory. However, the fractional block that is required to achieve these effects is unknown. Here, we measured the dose-dependent suppression of contextual memory in vivo by systemic administration of the competitive antagonist (R,S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP); in parallel, we measured the concentration-dependent block by CPP of NMDAR-mediated synapses and LTP of excitatory synapses in hippocampal brain slices in vitro. We found that the dose of CPP that suppresses contextual memory in vivo (EC50 = 2.3 mg/kg) corresponds to a free concentration of 53 nM. Surprisingly, applying this concentration of CPP to hippocampal brain slices had no effect on the NMDAR component of evoked field excitatory postsynaptic potentials (fEPSP_NMDA), or on LTP. Rather, the IC50 for blocking the fEPSP_NMDA was 434 nM, and for blocking LTP was 361 nM – both nearly an order of magnitude higher. We conclude that memory impairment produced by systemically administered CPP is not due primarily to its blockade of NMDARs on hippocampal pyramidal neurons. Rather, systemic CPP suppresses memory formation by actions elsewhere in the memory-encoding circuitry.
Human brain slices for epilepsy research: Pitfalls, solutions and future challenges
2016, Journal of Neuroscience Methods
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Thus, tissue obtained from these patients is advantageous from the point of view of probing potential mechanisms underlying pharmacoresistance but less so for gaining insight into the process of epileptogenesis in the human brain. Using approaches initially developed for rodent brain slices by McIlwain and others (Collingridge, 1995), the ability to prepare and maintain slices of epileptic human cortical tissue gives unprecedented access to human brain tissue for subsequent interrogation with a variety of tools. At the time of this publication, there have been approximately 110 novel papers reporting on the use of human brain slices in vitro.
Increasingly, neuroscientists are taking the opportunity to use live human tissue obtained from elective neurosurgical procedures for electrophysiological studies in vitro. Access to this valuable resource permits unique studies into the network dynamics that contribute to the generation of pathological electrical activity in the human epileptic brain. Whilst this approach has provided insights into the mechanistic features of electrophysiological patterns associated with human epilepsy, it is not without technical and methodological challenges. This review outlines the main difficulties associated with working with epileptic human brain slices from the point of collection, through the stages of preparation, storage and recording. Moreover, it outlines the limitations, in terms of the nature of epileptic activity that can be observed in such tissue, in particular, the rarity of spontaneous ictal discharges, we discuss manipulations that can be utilised to induce such activity. In addition to discussing conventional electrophysiological techniques that are routinely employed in epileptic human brain slices, we review how imaging and multielectrode array recordings could provide novel insights into the network dynamics of human epileptogenesis. Acute studies in human brain slices are ultimately limited by the lifetime of the tissue so overcoming this issue provides increased opportunity for information gain. We review the literature with respect to organotypic culture techniques that may hold the key to prolonging the viability of this material. A combination of long-term culture techniques, viral transduction approaches and electrophysiology in human brain slices promotes the possibility of large scale monitoring and manipulation of neuronal activity in epileptic microcircuits.
Analysis of acute brain slices by electron microscopy: A correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning
2015, Journal of Structural Biology
Citation Excerpt :
Despite the increased use of in vivo recordings on conscious animals to provide new insights into cellular neurophysiology, acute brain slices remain the tool of choice for patch clamp recording to analyse biological features such as ion channels and calcium flux (Khurana and Li, 2013). Since their introduction 50 years ago by Henry McIlwain (Collingridge, 1995; McIlwain, 1958), acute brain slices have been studied in detail by using advanced fluorescence microscopy techniques such as Stimulated Emission-Depletion (STED) microscopy, Stochastic Optical Reconstruction Microscopy (STORM) and Photoactivated Localization Microscopy (PALM), bringing the resolution under the micrometre level (Huang et al., 2010). Even more recently, a combination of two photon excitation and pulsed STED microscopy (Bethge et al., 2013) improved the imaging resolution on acute brain slices to a point-spread function of 62 nm for 40 nm fluorescent beads.
Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu’s protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.
An aerator for brain slice experiments in individual cell culture plate wells
2014, Journal of Neuroscience Methods
Citation Excerpt :
Ex vivo acute living brain slices are a broadly employed and powerful experimental approach (Collingridge, 1995; Khurana and Li, 2013; Li and McIlwain, 1957; McIlwain et al., 1951; Yamamoto and McIlwain, 1966).
Ex vivo acute living brain slices are a broadly employed and powerful experimental preparation. Most new technology regarding this tissue has involved the chamber used when performing electrophysiological experiments. Alternatively we instead focus on the creation of a simple, versatile aerator designed to allow maintenance and manipulation of acute brain slices and potentially other tissue in a multi-well cell culture plate.
Here we present an easily manufactured aerator designed to fit into a 24-well cell culture plate. It features a nylon mesh and a single microhole to enable gas delivery without compromising tissue stability. The aerator is designed to be individually controlled, allowing both high throughput and single well experiments.
The aerator was validated by testing material leach, dissolved oxygen delivery, brain slice viability and neuronal electrophysiology. Example experiments are also presented, including a test of whether β1-adrenergic receptor activation regulates gene expression in ex vivo dorsal striatum using qPCR.
Key differences include enhanced control over gas delivery to individual wells containing brain slices, decreased necessary volume, a sample restraint to reduce movement artifacts, the potential to be sterilized, the avoidance of materials that absorb water and small biological molecules, minimal production costs, and increased experimental throughput.
This new aerator is of high utility and will be useful for experiments involving brain slices and other potentially tissue samples in 24-well cell culture plates.
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Research paperThe brain slice preparation: a tribute to the pioneer Henry McIlwain

Abstract

Continuous recording of changes in membrane potential in mammalian cerebral tissues in vitro; recovery after depolarization by added substances

J. Physiol.

Maintenance of resting membrane potentials in slices of mammalian cerebral cortex and other tissues in vitro

J. Physiol.

Techniques in tissue metabolism. 1. A mechanical chopper

Biochem. J.

Research paper
The brain slice preparation: a tribute to the pioneer Henry McIlwain