NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: An in vitro model for the study of neural precursor cell proliferation

doi:10.1016/j.brainres.2010.05.024

Brain Research

Volume 1359, 4 November 2010, Pages 22-32

https://doi.org/10.1016/j.brainres.2010.05.024 Get rights and content

Abstract

We present a model for the study of injury-induced neurogenesis in the dentate gyrus (DG) in murine organotypic hippocampal slice cultures (OHCs). A brief exposure of 8-day-old hippocampal slice cultures to the glutamate receptor agonist N-methyl-d-aspartate (NMDA; 20–50 µM for 30 min) caused a selective excitotoxic injury in the CA1 subfield of the hippocampus that matured over a period of 24 h. The insult resulted in a prominent up-regulation of proliferating nuclei within the OHC dentate gyrus (DG), and a corresponding increase in Ki67/doublecortin double-positive cells in the SGZ of the dentate gyrus. 5-bromo-2-deoxyuridine (BrdU)-labelling of the OHCs for three days subsequent to the NMDA exposure revealed significantly increased BrdU incorporation within the DG (SGZ and GCL) of the hippocampus. Doublecortin immunofluorescence indicated a concurrent up-regulation of neuronal precursor cells specifically in the SGZ and GCL. Significantly increased BrdU incorporation could be detected up to 6–9 days after termination of the NMDA exposure. The model presented here enables easy manipulation and follow-up of injury-induced neuroblast proliferation in the DG that is amenable to the study of transgenic mice.

Introduction

Neurogenesis continues throughout adulthood within specific areas in the brain of rats, mice and humans (Altman and Das, 1965, Curtis et al., 2007, Kaplan and Hinds, 1977). One of these areas is the hippocampal dentate gyrus (DG; Cameron and McKay, 2001), where the neuronal progenitor cells reside and proliferate in the subgranular zone (SGZ), develop a neuronal phenotype (Kaplan and Hinds, 1977), and migrate into the granule cell layer (GCL; Altman and Bayer, 1990). After migration they adopt morphological characteristics of granule cells (Kaplan and Hinds, 1977) and extend axonal projections to the neurons of the CA3 region (Stanfield and Trice, 1988).

Animal studies have firmly established that insults to the brain such as cerebral ischemia, status epilepticus and traumatic brain injury result in increased neurogenesis in the DG following a latent period (Parent et al., 1997, Arvidsson et al., 2001, Kernie et al., 2001). Excessive release of the excitatory neurotransmitter glutamate and excitotoxic overactivation of glutamate receptors, in particular N-methyl-d-aspartate (NMDA) and kainic acid receptors, have been associated with these types of injuries (Choi, 1994, Besancon et al., 2008, Doyle et al., 2008, Henshall, 2007). There is evidence for a role of glutamate receptor activation in the stimulation of neurogenesis in the DG of naïve animals (Nacher et al., 2001, Nacher and McEwen, 2006, Nacher et al., 2007). Recent studies have demonstrated that treatment with glutamate receptor antagonists reduced neurogenesis but also decreased tissue damage in models of cerebral ischemia (Bernabeu and Sharp, 2000, Arvidsson et al., 2001) suggesting that injury-induced neurogenesis may be a consequence of an excitotoxic injury.

Organotypic hippocampal slice cultures (OHCs) have been widely used to study development, physiology, and injury of the hippocampal formation (De Roo et al., 2008, Murphy et al., 2008, Reid et al., 2008, Vornov et al., 1991). Unlike dissociated neuronal cultures, organotypic cultures retain a complex three-dimensional organisation of nervous tissue very similar to that in vivo, but are easily accessible to short- or long-term treatments, genetic manipulation, and short- or long-term evaluation of physiological parameters. These advantages make them attractive for investigating proliferation and development of newly generated granule cells under defined experimental conditions. Here, we have developed a model of excitotoxic injury-induced neural precursor cell proliferation in the dentate gyrus using murine OHCs.

Section snippets

A 30 min exposure of OHCs to NMDA leads to selective neuronal damage in the CA1 hippocampal subfield

In order to establish a reproducible model of excitotoxic injury in murine OHCs, 8-day-old slice cultures were exposed to increasing concentrations of NMDA (5–1000 µM) or buffer only for a period of 30 min. After 24 h, cell death was evaluated by propidium iodide (PI) uptake and slices were viewed under a fluorescence microscope (Murphy et al., 2008). While exposure to 5 µM NMDA did not produce any injury, exposure to 20 or 50 µM NMDA resulted in neuronal injury to the CA1 hippocampal subfield of

Discussion

In the present study, we have established an OHC model for the study of excitotoxic injury to the CA1 subfield and the subsequent proliferation of neuronal precursor cells in the DG. Analysis of propidium iodide (PI) fluorescence intensities within each of the hippocampal subfields after treatment with 20 and 50 µM NMDA revealed a pronounced injury that was restricted to the CA1-region, and this was paralleled by an increased incorporation of the proliferation marker BrdU into the dentate gyrus

Preparation and maintenance of organotypic hippocampal slice cultures (OHCs)

OHCs were prepared according to a previously described procedure with modifications (Kristensen et al., 2001). In brief, 10-day-old C57BL6 mice were decapitated; the brain was removed and hippocampi were dissected and transversely sliced into 350 µm thick slices on a McIllwain tissue chopper (Mickle Laboratory, Guldfort, UK). The slices were transferred into dissection medium containing Hank's balanced salt solution (Gibco, Biosciences, Dublin, Ireland), 20 mmol/l HEPES, 100 U/ml penicillin, 100

Acknowledgment

This research was supported by grants from Science Foundation Ireland to JHMP (03/RP1/B344; 08/IN1/1949).

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It is unlikely that upregulation of BrdU-positive cells in the CA1 region of the hippocampus after the NMDA treatment is due to an increase in neurogenesis since lines of evidence have indicated that the neurogenesis occurs at the dentate gyrus (DG) region of OHSCs (Bunk et al., 2010; Kamada et al., 2004). Moreover, previous reports have suggested that NMDA treatment can also trigger proliferation of astrocytes (Condorelli et al., 1989) or change of astrocytes to reactive types (Bunk et al., 2010; Kunkler and Kraig, 1997). In this study, we demonstrated that tacrine treatment down-regulated BrdU-positive cells and reduced neuronal cell damage on day 3 after NMDA pretreatment.
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¹: These authors contributed equally to this work.

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Research ReportNMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: An in vitro model for the study of neural precursor cell proliferation

Abstract

Introduction

Section snippets

A 30 min exposure of OHCs to NMDA leads to selective neuronal damage in the CA1 hippocampal subfield

Discussion

Preparation and maintenance of organotypic hippocampal slice cultures (OHCs)

Acknowledgment

Neuron

Trends Pharmacol. Sci.

Neurobiol. Dis.

Prog. Brain Res.

Neuroscience

Neuropharmacology

Neuron

Exp. Neurol.

J. Neurosci. Methods

Trends Neurosci.

Brain Res.

J. Biol. Chem.

Neuroscience

Cell

Mol. Cell. Neurosci.

J. Neurosci. Methods

Exp. Neurol.

Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods

J. Comp. Neurol.

Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats

J. Comp. Neurol.

N-methyl-d-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke

Eur. J. Neurosci.

NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus

J. Cereb. Blood Flow Metab.

Transient expression of doublecortin during adult neurogenesis

J. Comp. Neurol.

Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus

J. Comp. Neurol.

NMDA receptor-mediated excitotoxic neuronal apoptosis in vitro and in vivo occurs in an ER stress and PUMA independent manner

J. Neurochem.

Activation of excitatory amino acid receptors reduces thymidine incorporation and cell proliferation rate in primary cultures of astrocytes

Glia

Research Report
NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: An in vitro model for the study of neural precursor cell proliferation