Neurotrophin Protection Against Toxicity Induced by Low Potassium and Nitroprusside in Cultured Cerebellar Granule Neurons

Abstract

Long‐term survival of cultured rat cerebellar granule neurons requires depolarizing concentrations of potassium (high potassium; 25 mM KCl). A high‐potassium culturing condition has been reported to increase the intracellular calcium concentration ([Ca2+]i) and the expression of brain‐derived neurotrophic factor (BDNF), which in turn induces the expression of neurotrophin‐3 (NT‐3) in these neurons. We therefore examined the neurotrophic effect of these two neurotrophins in low‐potassium (5 mM) cultures and their neuroprotective capabilities against sodium nitroprusside‐induced neurotoxicity in both low‐ and high‐potassium cultures. Neuronal survival and neurotrophic effects were monitored by [3H]ouabain binding and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays. In low‐potassium cultures, the neurotrophic effect of BDNF approached that found in high‐potassium cultures but was much more robust than that of NT‐3. In contrast, undifferentiated neurons cultured in high‐potassium medium were much less responsive to BDNF and not responsive at all to NT‐3. Induction of nitroprusside neurotoxicity occurred more readily in low‐ than in high‐potassium cultures. BDNF, NT‐3, and a high potassium concentration, alone or in combination, were unable to protect neurons treated with nitroprusside at 50 or 100 µM. However, the neurotoxicity of a lower dose of nitroprusside (10 µM) was reversed by the combined actions of these two neurotrophins in low‐potassium cultures and by BDNF alone in high‐potassium cultures. Because nitroprusside neurotoxicity is less robust in high‐potassium cultures, high‐potassium‐induced BDNF expression and subsequent NT‐3 expression may participate in its neuroprotection and neurotrophism in these cultures. Also, we found that toxic doses of nitroprusside antagonized KCl‐ and NMDA‐induced rises in [Ca2+]i, suggesting that this effect is related to nitroprusside‐induced neurotoxicity.

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“…NT‐3 is a neurotrophic factor that has been extensively studied for its neuroprotective potential, and even though its mechanism of action is not fully understood it has been suggested that NT‐3 exerts direct effects on neurons subjected to oxidative stress or exposed to neurotoxic substances (Yu and Chuang, ; Korsak et al, ; Ma et al, ). Inflammation, hypoxia, and excitotoxicity are conditions likely to be present in SCSC following culture preparation.…”

“…Neurotrophin (NT)‐3 is a neurotrophic factor that has been extensively studied for its neuroprotective effects (Ma et al, ; Yu and Chuang, ). Briefly, NT‐3 enhances neuronal survival of mechanically injured neurons in vitro (Ma et al, ) and improves neurological recovery followed ischemic brain damage in vivo (Zhang et al, ).…”