Time-dependent changes in BDNF expression of pentylenetetrazole-induced hippocampal astrocytes in vitro

doi:10.1016/j.brainres.2011.12.035

Brain Research

Volume 1439, 23 February 2012, Pages 1-6

https://doi.org/10.1016/j.brainres.2011.12.035 Get rights and content

Abstract

Pentylenetetrazole (PTZ), a γ-aminobutyric acid (GABA_A) receptor antagonist, has been used extensively to induce seizures in animal models of epilepsy. The aim of the present study was to investigate the effects of PTZ on hippocampal astrocytes. Cells were incubated with 10, 20, or 40 mM PTZ for 24 h and viability and apoptosis were examined using an MTT assay and Hoechst staining. The high concentration of PTZ (20 and 40 mM) resulted in a significant decrease in viability (MTT: 83.6 ± 7.8% and 69.3 ± 4.2%, respectively) (P < 0.01), whereas the lower concentration of PTZ (10 mM) did not induce cell apoptosis or reduce viability. When cells were treated with 10 mM PTZ for 0, 0.5, 2, 4, 8, 12, or 24 h, the level of brain-derived neurotrophic factor (BDNF), both protein and mRNA, was significantly reduced at 2 to 12 h of culture (P < 0.01), with maximal reduction detected at 8 h; expression was restored to near control levels after 24 h. Collectively, our results suggest that astrocytes may participate in epilepsy through a marked, but transient decrease in BDNF expression.

Highlights

► PTZ treatment reduces astrocyte viability in a dose-dependent manner. ► PTZ induces BDNF expression in astrocytes in a time-dependent manner. ► PTZ induces time-dependent changes in the expression of BDNF mRNA in astrocytes.

Introduction

The central nervous system (CNS) consists of two types of cells, neurons and glia cells, and their bidirectional communication system can be critical in modulating integration within the nervous system. Astrocytes are, in fact, an integral element of the circuitry for synaptic plasticity and play important physiological roles in the CNS, such as alteration of synaptic transmission and synchronization of neuronal firing (Binder and Steinhäuser, 2006, Seifert et al., 2010). Furthermore, astrocytes can synthesize and secrete a large variety of neurotrophic factors including brain-derived neurotrophic factor (BDNF) (Cardile et al., 2003).

BDNF is a small dimeric protein that belongs to the neurotrophin family of structurally related proteins and was originally identified as a crucial neuronal survival factor (Barde et al., 1982). BDNF is abundant in the brain and, in addition to promoting the proliferation and differentiation of neurons, it influences the shape and number of dendritic spines, impacting the morphological development of neurons (Cohen-Cory et al., 2010, Kuczewski et al., 2010). Increasing evidence indicates that BDNF may participate in synaptic plasticity and in processes such as learning, memory, and age-related memory deficits (Kuczewski et al., 2010, Pillai, 2008, Yamada and Nabeshima, 2003).

Pentylenetetrazole (PTZ) is a cerebral stimulant that antagonizes the γ-aminobutyric acid type A receptor (GABA_A) that interacts with the picrotoxin-barbiturate binding site, inhibiting GABA_A receptors, closing Cl⁻ channels, and provoking seizures (Qu et al., 2005). GABA_A receptors are present on glial cells in the brain (Verkhratsky and Steinhäuser, 2000) and recent studies demonstrated that expression of glial fibrillary acidic protein (GFAP) is increased in rat hippocampus by acute convulsive PTZ challenge (Gurses et al., 2009). Additionally, PTZ affects mitochondrial metabolism and glycolysis in cortical and cerebellar astrocytes during short- or long-term culture (Qu et al., 2005). However, the effects of PTZ on astrocyte-derived BDNF expression remain unclear. Therefore, in the current study, we evaluated cell viability and BDNF expression in hippocampal astrocytes cultured in the presence of PTZ.

Section snippets

PTZ treatment reduces astrocyte viability in a dose-dependent manner

After a 24 h incubation, the high concentrations of PTZ (20 and 40 mM) promoted a significant decrease in astrocyte viability as determined using an MTT assay (MTT: 83.6 ± 7.8 and 69.3 ± 4.2%, respectively) (P < 0.01); in contrast, astrocyte cultures exposed to the 10 mM PTZ did not display reduced viability (MTT: 92.4 ± 7.6%) (P > 0.05) (Fig.1). Thus, there was a dose-dependent PTZ-mediated decrease in astrocyte viability.

No apoptosis was induced by astrocyte exposure to a low concentration of PTZ

Hoechst 33342 is a bis-benzimidazole dye that penetrates the plasma membrane and

Discussion

In the current study, we demonstrated that exposure to PTZ resulted in a dose-dependent decrease in astrocyte viability. PTZ is a chemical convulsant that binds to the picrotoxin-binding site of the post-synaptic GABA_A receptor and suppresses the inhibitory effects of some neurotransmitters, especially GABA. Injection of sub-convulsant doses of PTZ (< 40 mg/kg) induces absence-like seizures (Snead et al., 2000); whereas injection of 70 mg PTZ/kg body weight results in the typical generalized tonic

Conclusion

Our findings provide direct evidence that time-dependent changes in BDNF mRNA and protein expression occur in hippocampal astrocytes when treated with a concentration of PTZ that induces an ictal-like epileptiform activity but does not promote astrocyte apoptosis. Collectively, these findings suggest that astrocytes may play some role in epilepsy. Additional studies are currently being performed at our institute to further define their involvement.

Primary astrocyte cultures

Astrocyte cultures were prepared from the hippocampus of 2-day-old neonatal Sprague–Dawley rats following mechanical dissociation as described previously (McCarthy and De Vellis, 1980). Briefly, dissociated cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM)-Ham's F-12 Culture Medium (Gibco, Invitrogen. Corporation, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) and 1 mM glutamine (Gibco). Cells were then cultured in an uncoated 25-cm² flask at a density of

Acknowledgments

This work was supported by Shanghai Leading Academic Discipline Project (Number: S30201, S30205), Shanghai Science and Technology Foundation of China (Number: 1052nm04200), the International Cooperation projects of Shanghai Science and Technology Committee (Number: 09410706200) and Shanghai Education Committee.

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¹: Authors contributed equally to this work.

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Research ReportTime-dependent changes in BDNF expression of pentylenetetrazole-induced hippocampal astrocytes in vitro

Abstract

Highlights

Introduction

Section snippets

PTZ treatment reduces astrocyte viability in a dose-dependent manner

No apoptosis was induced by astrocyte exposure to a low concentration of PTZ

Discussion

Conclusion

Primary astrocyte cultures

Acknowledgments

Neuroscience

Trends Neurosci.

Brain Res.

Neurochem. Int.

Brain Res.

Neurosci. Lett.

Brain Res.

Neurosci. Lett.

Trends Neurosci.

Brain Res. Rev.

Brain Res. Brain Res. Rev.

J. Pharmacol. Sci.

Research Report
Time-dependent changes in BDNF expression of pentylenetetrazole-induced hippocampal astrocytes in vitro