Validation of a neocortical slice preparation for the study of epileptiform activity

doi:10.1016/0165-0270(88)90005-2

Journal of Neuroscience Methods

Volume 23, Issue 3, April 1988, Pages 211-224

https://doi.org/10.1016/0165-0270(88)90005-2 Get rights and content

Abstract

A simple slice chamber was designed to achieve easy manipulations of temperature, ionic composition and drug concentrations. Spontaneous and evoked extracellular potentials could be recorded with glass microelectrodes from 500 μm thick slices of rat frontal neocortex. In the absence of magnesium ions in the superfusing medium or in the presence of convulsant agents, epileptiform activity was seen. The amplitude of this activity was greatest in layer II/III, each burst consisting of a long-lasting negative potential on the decay phase of which were superimposed many afterpotentials. There were multiple foci from which spontaneous epileptiform bursts spread to other ipsi- and contralateral parts of the cortex via both the grey and white matter. Although such bursts were observed between 23 and 37° C, optimal recording of discrete epileptiform activity was achieved at 29 ± 1° C. Decreasing extracellular calcium or increasing extracellular concentrations of potassium enhanced burst discharges. Proconvulsant agents initiated both interictal and ictal epileptiform events. This, together with the reduction of epileptiform activity by standard anticonvulsant drugs such as carbamazepine and phenobarbitone suggested that this in vitro model may be useful for studying the pharmacology of epileptogenesis and for developing new therapeutic strategies for epilepsy.

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Despite the prevalent use of the ex vivo brain slice preparation in neurophysiology research, a reliable method for judging tissue viability ― and thus suitability of a slice for inclusion in an experiment ― is lacking. The utility of indirect electrophysiological measures of tissue health is model-specific and needs to be used cautiously. In this study, we verify a more direct test of slice viability, based on tissue oxygen consumption rate.
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Following tissue preparation, the slices were transferred to a perfusion recording bath and maintained under submerged conditions in no-Mg aCSF. Exposure of cortical slices to no-Mg aCSF activates the tissue by unblocking NMDA receptors (Aram and Lodge, 1988), resulting in the generation of repeating, spontaneous paroxysmal events known as seizure-like events (SLEs). SLEs are recordable from all cortical layers and regions and reflect the coordinated firing of mixed populations of excitatory and inhibitory neurons.
Hypoxic brain injury is a leading cause of loss of quality of life globally for which there are currently no effective treatments. There has been increasing interest in incorporating photosynthesising agents into hypoxic tissue as a mechanism for in situ oxygen delivery, independent of vascular perfusion. To date this has not been tested in the brain.
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Subsequently, the slices were immersed in no-Mg aCSF at room temperature (approximately 21 °C) for at least 60 min. Exposure to no-Mg aCSF activates the tissue by unblocking NMDA receptors (Aram and Lodge, 1988), resulting in the generation of repeating, spontaneous paroxysmal events commonly known as seizure-like events (SLEs). SLE activity is sensitive to hypoxic challenge (Voss et al., 2020) and provides a relatively non-invasive electrophysiological method for quantifying tissue viability over time (Voss et al., 2013).
Hypoxic-ischaemic brain injury is a major cause of morbidity and mortality internationally. Using an in vitro isolated cortex model, this study investigated the optimal cerebrospinal fluid oxygenation parameters for rescuing metabolically challenged cortical tissue. In particular, we asked whether maximizing oxygen content with oxygen nanobubbles could support improved tissue recovery. Mouse cortical slices were metabolically starved, followed by recovery in artificial cerebrospinal fluid (aCSF) containing different levels of dissolved oxygen ranging from mean(SD) 2(0.5) to 39(1.0) mg/L; with and without oxygen nanobubbles. Tissue recovery was assessed by quantifying and comparing the amplitude, length, high frequency content and event frequency of seizure-like events generated in no-magnesium aCSF at the beginning and end of the protocol. In general, there was improved recovery with increasing oxygen content up to 25–34 mg/L. The outcome of slices recovered in nanobubbled aCSF was no different to conventionally oxygenated slices with similar dissolved oxygen content. Dissolved oxygen content above 34 mg/L afforded no additional benefit. In conclusion, aCSF dissolved oxygen content of approximately 30 mg/L is optimal for cortical tissue recovery from metabolic starvation, which is easily achievable using conventional oxygenation methods. Oxygen in the form of nanobubbles does not appear to be readily available for tissue oxidative processes in this model.
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Subsequently the slices were immersed in no-Mg aCSF at room temperature (approximately 21 °C) for at least 60 min. Exposure to no-Mg aCSF activates the tissue by unblocking NMDA receptors (Aram and Lodge, 1988), resulting in the generation of repeating, spontaneous paroxysmal events commonly known as seizure-like events (SLEs). The slices were transferred one at a time to a submersion-style perfusion bath (Kerr Scientific Instruments, Dunedin, NZ) which was continuously flowed with the aCSF solution of choice, at an adjustable flow rate.
The in vitro cortical slice preparation is a widely used tool for electrophysiological investigation of brain neurophysiology. However, slice quality can be highly variable despite attempts to standardise practice. The purpose of this study was to determine the extent to which this variability is due to sensitivity to aspects of the preparation methodology.
The study ultilised the no-magnesium seizure-like event (SLE) model and was in two parts. In the first, slice outcome was quantified following a standardised hypoxic-ischaemic insult applied to pre-prepared slices. The hypoxia-induced changes in SLE activity were used to characterise the effect of tissue damage on commonly measured electrophysiological variables. In the second, multivariate data associated with the slice preparation methodology was collected and related to tissue outcome, according to the same SLE characteristics.
Hypoxia-ischaemia damage was reflected most strongly in a reduction in SLE amplitude, inter-event frequency and intra-event high frequency activity. The number of active locations was also markedly reduced. In the second part of the study, between 21% and 47% of outcome variation was attributable to variability in the methodological parameters tested. Most important were slice position from the tissue block, depth of slice submersion during recording and the experience level of the experimenter. The latter showed a paradoxical relationship between inexperience, heightened SLE amplitude and reduced spatial viability.
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Subsequently the slices were immersed in aCSF void of magnesium (no-Mg aCSF) at room temperature (approximately 21 °C) for at least 60 minutes. Exposure to no-Mg aCSF activates the tissue by unblocking NMDA receptors [6], resulting in the generation of repeating, spontaneous paroxysmal events known as seizure-like events (SLEs) [7]. Normal aCSF was composed of 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 1.25 mM NaH2PO4, 2.5 mM NaHCO3, 10 mM HEPES and 10 mM D-glucose.
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In sharp contrast, the dynamics of the biochemical components in endfeet under (patho)physiological conditions are poorly understood. In this study, we utilized the well characterized zero magnesium (0 Mg2+) brain slice model of epilepsy, which induces epileptiform discharges in wide areas of the neocortex (15–17), as a model system to examine the effect of (patho)physiological neuronal activities on the biochemical organizations of endfeet in cortical astrocytes. Here, we report that upon the epileptic activation of neurons, βDG is down-regulated with distinct kinetics from the pathological activation of neurons and with a concurrent dysfunction of the astrocytic endfeet.
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Research paperValidation of a neocortical slice preparation for the study of epileptiform activity

Abstract

Brain Res.

Prog. Neurobiol.

Brain Res.

Brain Res.

Neurosci. Lett.

Eur. J. Pharmacol.

Exp. Neurol.

Prog. Neurobiol.

Neuroscience

Brain Res.

Lancet

Epilepsy Res.

Neuroscience

Epileptiform burst afterhyperpolarization: calcium-dependent potassium potential in hippocampal CAI pyramidal cells

Science

Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro

J. Physiol. (Lond.)

N-Methylaspartate antagonists block epileptiform discharges in rat cortical slices maintained in magnesium free medium

Br. J. Pharmacol.

Effects of anticonvulsants on NMA receptor-mediated epileptiform activity in rat cortical slices perfused with magnesium-free CSF

Br. J. Pharmacol.

Threshold effects of N-methyl-d-aspartate (NMDA) and 2-amino-5-phosphonovaleric acid (2 APV) on the spontaneous activity of neocortical single neurones in the urethane anaesthetised rat

Exp. Brain Res.

Selective depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinal cord preparations

J. Physiol. (Lond.)

Initiation of synchronized neuronal bursting in neocortex

Nature (Lond.)

Cellular mechanisms of neocortical epileptogenesis in an acute experimental model

N-Methyl-d-aspartate activates voltage-dependent calcium conductance in rat hippocampal pyramidal cells

J. Physiol. (Lond.)

Allosteric effects of Mg2+ on the gating of Ca2+-activated K+ channels from mammalian skeletal muscle

J. Exp. Biol.

Research paper
Validation of a neocortical slice preparation for the study of epileptiform activity

Threshold effects of $N- methyl- d -aspartate$ (NMDA) and 2-amino-5-phosphonovaleric acid (2 APV) on the spontaneous activity of neocortical single neurones in the urethane anaesthetised rat

$N- Methyl- d -aspartate$ activates voltage-dependent calcium conductance in rat hippocampal pyramidal cells

Allosteric effects of Mg²⁺ on the gating of Ca²⁺-activated K⁺ channels from mammalian skeletal muscle