Dopamine protects neurons against glutamate-induced excitotoxicity

Abstract

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.

Figure 1

DA modulates the effect of low physiological concentrations of glutamate on [Ca²⁺]_c. Short application of 5 μM glutamate to hippocampal neurons induced a rise in [Ca²⁺]_c (Fura-2 ratio) (a). (b) 5 μM DA significantly reduced glutamate-induced calcium signal. (c) Effect of different concentrations of DA on the glutamate induced Ca²⁺ rise in hippocampal neurons. *P<0.05, **P<0.001

Figure 2

Changes in the [Ca²⁺]_c response to toxic concentrations of glutamate. Kinetic measurements of changes in [Ca²⁺]_c (Fura-FF ratio) from single neurons in response to 100 μM glutamate in midbrain (a) and cortical (b) cultures. Glutamate (100 μM) and glycine (10 μM) were applied in a Mg²⁺-free solution. (c) Pretreatment (5 min) of cortical neurons with 0.5 μM DA inhibits appearance of DCD in response to glutamate. Histogram showing the time from the application of glutamate until the start of the appearance of DCD in neurons from different brain regions in response to 100 μM glutamate (+10 μM glycine in Mg²⁺-free) with varying concentrations of DA (d). *P<0.05, **P<0.001. (e) Co-application of 20 μM selegiline and 0.5 μM DA 5 min before the experiment completely inhibited the glutamate-induced DCD. (f) Preincubation (5 min) with selegiline did not prevent the glutamate-induced DCD

Figure 3

Effect of DA and DA antagonists on glutamate-induced [Ca²⁺]_c changes in primary neurons. (a) 0.5 μM DA inhibits appearance of DCD in midbrain neurons in response to 100 μM glutamate(+10 μM glycine in Mg²⁺-free). Preincubation (5 min) of 20 μM sulpiride (b) or 20 μM SCH-23390 (c) eliminates protective effect of DA against DCD. (d) The effect of 0.5 μM DA alone and in combination with 20 μM sulpiride or 20 μM SCH-23390 on time of appearance of glutamate-induced DCD. Histogram represent the time taken from application of 100 μM glutamate to the beginning of the DCD in neurons from the different brain region. **P<0.001

Figure 4

Effect of DA agonists on glutamate-induced [Ca²⁺]_c changes in primary neurons. (a) 100 μM glutamate increase [Ca²⁺]_c in primary hippocampal neurons. Pretreatment (5 min) of hippocampal neurons with 10 μM quinprole (b) or 10 μM chloro-APB (c) changed the shape of the glutamate-induced Ca²⁺ signal

Figure 5

Dependence of glutamate-induced neuronal cell death on the presence of DA. (a) The viability of primary cortical and midbrain neurons was measured 24 h after a 15 min application of 100 μM glutamate in 10 μM glycine, Mg²⁺-free medium, in the presence of either 0, 1, 10 or 20 μM DA, using PI fluorescence. Dead cells were counted with respect to total number of cells present, identified by staining nuclei with Hoechst 33342. (b) Cell death was further measured in cortical neurons after cells were pretreated for 10 min with either DA (1 μM) alone or in combination with 20 μM SCH-23390 or 20 μM sulpiride before application of 100 μM glutamate. DA and the inhibitors were left in the medium during the 15 min glutamate exposure. **P<0.05, *P<0.001; n.s., not significant