Attenuation of cell death mediated by membrane depolarization different from that by exogenous BDNF in cultured mouse cerebellar granule cells

doi:10.1016/S0169-328X(98)00062-X

Molecular Brain Research

Volume 56, Issues 1–2, May 1998, Pages 218-226

https://doi.org/10.1016/S0169-328X(98)00062-X Get rights and content

Abstract

Membrane depolarization accompanying calcium (Ca²⁺) influx into neurons is thought to play an essential role in controlling the survival and death of cultured mouse cerebellar granule cells (CGCs). In this study, we sequentially controlled the survival and death of CGCs in culture and monitored the expression of several kinds of genes including brain-derived neurotrophic factor (BDNF) gene. Deprivation and subsequent induction of membrane depolarization by lowering and re-elevating the extracellular concentration of potassium chloride, respectively, led to death of CGCs and then to an attenuation of the death process depending upon the Ca²⁺ influx into CGCs through voltage-dependent calcium channels (VDCCs). De novo protein synthesis was critical for attenuating the death of non-depolarized CGCs. Accompanying this attenuation was an activation of c-fos and BDNF genes and an inactivation of c-jun and neurotrophin-3 (NT-3) genes. The attenuation of cell death mediated by exogenous BDNF was only partial compared to that by membrane depolarization, suggesting that not only BDNF but also other factors could be involved in the membrane depolarization-mediated attenuation of death of CGCs. In good agreement with this observation, the mode of activation of c-fos, c-jun, BDNF and NT-3 genes induced by exogenous BDNF was different from that induced by membrane depolarization. Thus, membrane depolarization effectively attenuates the death of non-depolarized CGCs, the mode of which seems to be different from that mediated by BDNF alone.

Introduction

During the early development of rodent cerebellum, the cerebellar granule cells (CGCs) proliferate, migrate and differentiate, resulting in the formation of cerebellar cortexes. In this period, neuronal death of CGCs also occurs, amounting to 20–30% of the initial population [27]. The CGCs can successfully be maintained in culture, and their survival is dependent upon the membrane depolarization induced by elevating the potassium chloride (KCl) concentration in the medium 4, 7. Deprivation of membrane depolarization by lowering the KCl concentration of the medium, however, is known to lead to apoptotic death of CGCs 5, 6. Thus, survival and death of CGCs are largely controlled by the depolarized states of cells, which affect the Ca²⁺ influx into neurons through voltage-dependent calcium channels (VDCCs).

The survival of CGCs in culture is also controlled by exogenously added brain-derived neurotrophic factor (BDNF) 8, 13, 15. BDNF is a neurotrophin thought to be involved in controlling the survival and differentiation of selective populations of neurons in the peripheral and central nervous systems during embryonic development 16, 20. In primary culture of rat hippocampal or cerebral cortex neurons, it has already been demonstrated that BDNF mRNA expression can be induced by membrane depolarization [28]and endogenously synthesized BDNF may affect the survival of neurons [9]. Since CGCs also express BDNF and Trk-B [15], a BDNF-specific receptor, it can be speculated that BDNF endogenously synthesized in response to the depolarization of cell membrane is involved in the survival of CGCs, probably in an autocrine fashion, as already demonstrated with sensory neurons of the dorsal root ganglion [1]. However, it is still unknown to what degree BDNF accounts for the survival of CGCs mediated by membrane depolarization and, furthermore, whether the survival of CGCs controlled by membrane depolarization and BDNF are mediated by the same mechanisms or not.

Apoptotic cell death is observed in a variety of neurons during the development of the peripheral nervous system, and several cellular steps involving the expression of death or survival-related genes have been documented in the apoptosis of neurons [11]. Potassium-deprived apoptosis of CGCs in culture is also dependent upon de novo mRNA and protein synthesis [23], and can be blocked by high potassium [6], the mechanism of which has not yet been elucidated. In the present study, therefore, we sequentially controlled the death and survival of CGCs in culture by changing the extracellular KCl concentration and, then, compared the levels of cell survival and the patterns of mRNA expression of several kinds of genes including BDNF gene. In the present paper, we found that membrane depolarization protected the non-depolarized CGCs from death more effectively than exogenous BDNF with a different pattern of gene expression.

Section snippets

Reagents

Nicardipine and cycloheximide were purchased from Sigma. BDNF was generously donated by Sumitomo Seiyaku, Japan.

Cell culture and treatment

A primary culture of mouse cerebellar granule cells was prepared from 1-week-old mice (ICR). The cerebella were excised and cut with scissors into small pieces, collected by centrifugation at 800×g for 5 min, then incubated with 0.5 ml of phosphate-buffered saline (PBS⁽⁻⁾) containing 0.125% trypsin (GIBCO) and 1 mM EDTA for 15 min at 37°C. Approximately 4 ml of Dulbecco's modified

Attenuation of cell death following re-elevation of KCl concentration

After maintaining cultured mouse cerebellar granule cells (CGCs) in 25 mM KCl for 4 days, we replaced the medium with that containing 5, 12.5 and 25 mM KCl, respectively, and then cultured the cells for another 24 h to measure LDH release into the culturing medium (Fig. 1A). As the KCl concentration decreased, the LDH activities in the medium increased. In parallel to the increase in LDH release, microscopic observations of living cells stained by fluorescein diacetate (FDA) indicated that the

Discussion

It has been well established that an elevation of KCl concentration in culture medium ensures the survival of CGCs 4, 7, 14. As shown in Fig. 1A, the higher the extracellular KCl concentration, the lower the LDH release into the medium, indicating that the high potassium concentration in the medium supports the survival of CGCs in culture. Deprivation of membrane depolarization by lowering the extracellular KCl concentration from 25 to 5 mM, however, induced neuronal death of CGCs in culture.

Acknowledgements

We wish to thank Sumitomo Seiyaku Japan for donating BDNF. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan.

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Citation Excerpt :
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Research reportAttenuation of cell death mediated by membrane depolarization different from that by exogenous BDNF in cultured mouse cerebellar granule cells

Abstract

Introduction

Section snippets

Reagents

Cell culture and treatment

Attenuation of cell death following re-elevation of KCl concentration

Discussion

Acknowledgements

Neuron

Neuron

Cell

Dev. Brain Res.

Brain Res.

Trends Neurosci.

Neuron

Neuron

Brain Res.

Mol. Brain Res.

Biochem. Biophys. Res. Commun.

Neuron

Neurochem. Int.

Neuron

Research report
Attenuation of cell death mediated by membrane depolarization different from that by exogenous BDNF in cultured mouse cerebellar granule cells