Extending the Life Span of Acute Neuronal Tissue for Imaging and Electrophysiological Studies

Buskila, Yossi; Bellot-Saez, Alba; Kékesi, Orsolya; Cameron, Morven A.; Morley, John W.

doi:10.1007/978-1-4939-9944-6_10

Cited by 11 publications

(10 citation statements)

References 73 publications

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“…Excitotoxicity as a cause of injury in acute slices was reported initially by Feig and Lipton (Feig and Lipton, 1990 ), and it has been invoked in many subsequent studies as a reason for using glutamate receptor antagonists during slice preparation to block injury and promote recovery (Buskila et al, 2020 ). Feig and Lipton showed that morphological evidence of injury in guinea pig slices, in particular swelling of neuronal cell bodies, was alleviated by inclusion of ketamine, an NMDA receptor blocker in the recovery medium, or using a medium lacking calcium but with high magnesium (10 mM).…”

Section: Discussionmentioning

confidence: 99%

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus

Rimmele

Andersen

et al. 2021

Front. Cell. Neurosci.

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GLT-1, the major glutamate transporter in the mammalian central nervous system, is expressed in presynaptic terminals that use glutamate as a neurotransmitter, in addition to astrocytes. It is widely assumed that glutamate homeostasis is regulated primarily by glutamate transporters expressed in astrocytes, leaving the function of GLT-1 in neurons relatively unexplored. We generated conditional GLT-1 knockout (KO) mouse lines to understand the cell-specific functions of GLT-1. We found that stimulus-evoked field extracellular postsynaptic potentials (fEPSPs) recorded in the CA1 region of the hippocampus were normal in the astrocytic GLT-1 KO but were reduced and often absent in the neuronal GLT-1 KO at 40 weeks. The failure of fEPSP generation in the neuronal GLT-1 KO was also observed in slices from 20 weeks old mice but not consistently from 10 weeks old mice. Using an extracellular FRET-based glutamate sensor, we found no difference in stimulus-evoked glutamate accumulation in the neuronal GLT-1 KO, suggesting a postsynaptic cause of the transmission failure. We hypothesized that excitotoxicity underlies the failure of functional recovery of slices from the neuronal GLT-1 KO. Consistent with this hypothesis, the non-competitive NMDA receptor antagonist MK801, when present in the ACSF during the recovery period following cutting of slices, promoted full restoration of fEPSP generation. The inclusion of an enzymatic glutamate scavenging system in the ACSF conferred partial protection. Excitotoxicity might be due to excess release or accumulation of excitatory amino acids, or to metabolic perturbation resulting in increased vulnerability to NMDA receptor activation. Previous studies have demonstrated a defect in the utilization of glutamate by synaptic mitochondria and aspartate production in the synGLT-1 KO in vivo, and we found evidence for similar metabolic perturbations in the slice preparation. In addition, mitochondrial cristae density was higher in synaptic mitochondria in the CA1 region in 20–25 weeks old synGLT-1 KO mice in the CA1 region, suggesting compensation for loss of axon terminal GLT-1 by increased mitochondrial efficiency. These data suggest that GLT-1 expressed in presynaptic terminals serves an important role in the regulation of vulnerability to excitotoxicity, and this regulation may be related to the metabolic role of GLT-1 expressed in glutamatergic axon terminals.

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Section: Discussionmentioning

confidence: 99%

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus

Rimmele

Andersen

et al. 2021

Front. Cell. Neurosci.

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“…The Braincubator is an incubation system that closely monitors and controls pH, carbogen flow and temperature, as well as irradiating bacteria through a separate UV chamber [ 90 , 91 ]. Slices were initially incubated for 12 min at 35 °C, after which they were allowed to cool to 15–16 °C and kept in the Braincubator TM for at least 30 min before any measurements [ 92 , 93 ].…”

Section: Methodsmentioning

confidence: 99%

Neuromodulation of Astrocytic K+ Clearance

Bellot-Saez

Stevenson

Kékesi

et al. 2021

IJMS

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Potassium homeostasis is fundamental for brain function. Therefore, effective removal of excessive K+ from the synaptic cleft during neuronal activity is paramount. Astrocytes play a key role in K+ clearance from the extracellular milieu using various mechanisms, including uptake via Kir channels and the Na+-K+ ATPase, and spatial buffering through the astrocytic gap-junction coupled network. Recently we showed that alterations in the concentrations of extracellular potassium ([K+]o) or impairments of the astrocytic clearance mechanism affect the resonance and oscillatory behavior of both the individual and networks of neurons. These results indicate that astrocytes have the potential to modulate neuronal network activity, however, the cellular effectors that may affect the astrocytic K+ clearance process are still unknown. In this study, we have investigated the impact of neuromodulators, which are known to mediate changes in network oscillatory behavior, on the astrocytic clearance process. Our results suggest that while some neuromodulators (5-HT; NA) might affect astrocytic spatial buffering via gap-junctions, others (DA; Histamine) primarily affect the uptake mechanism via Kir channels. These results suggest that neuromodulators can affect network oscillatory activity through parallel activation of both neurons and astrocytes, establishing a synergistic mechanism to maximize the synchronous network activity.

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“…The brain slicing itself is a disruptive procedure that shatters the cell membranes through the slicing region. Therefore, the viable cells that remain confined between two layers of damaged tissue start to seal their membranes in order to avoid the spillover of cellular content originating from the adjacent damaged tissue [50]. Consequently, it can be hypothesized that the increase in the levels of all GPLs and sphingolipid classes found within 2.5h after brain slicing may be related to an upregulation in lipid biosynthesis inducing cell membrane rearrangements to reduce cellular leakage, followed by a stabilization of the tissue within the period between 2.5 and 5.5h after slicing.…”

Section: Resultsmentioning

confidence: 99%

Metabolic changes in brain slices over time: a multiplatform metabolomics approach

González-Riaño

Tapia-González

Perea

et al. 2020

Preprint

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Brain slice preparations are widely used for research in neuroscience. However, a high-quality preparation is essential and there is no consensus regarding stable parameters that can be used to define the status of the brain slice preparation after its collection at different time points. Thus, it is critical to establish the best experimental conditions for ex-vivo studies using brain slices for electrophysiological recording. In this study, we used a multiplatform (LC-MS and GC-MS) untargeted metabolomics-based approach to shed light on the metabolome and lipidome changes induced by the brain slice preparation process. We have found significant modifications in the levels of 300 compounds, including several lipid classes and their derivatives, as well as metabolites involved in the GABAergic pathway and the TCA cycle. All these preparation-dependent changes in the brain biochemistry should be taken into consideration for future studies to facilitate non-biased interpretations of the experimental results.

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Extending the Life Span of Acute Neuronal Tissue for Imaging and Electrophysiological Studies

Cited by 11 publications

References 73 publications

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus

Neuromodulation of Astrocytic K+ Clearance

Metabolic changes in brain slices over time: a multiplatform metabolomics approach

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