Relationship between NMDA receptor expression and MPP⁺ toxicity in cultured dopaminergic cells

Abstract

It has been suggested that excitotoxicity could be contributing to dopamine cell loss after methylphenylpyridinium ion (MPP+) exposure, although the literature regarding this is contradictory. Given that in cell culture excitotoxicity has been reported to be dependent on culture age, we postulated that these discrepant results might be explained by a difference in developmental expression of N-methyl-D-aspartate (NMDA) receptors. To test this, mesencephalic cells were cultured and the number of dopaminergic neurons (tyrosine hydroxylase-immunoreactive cells [TH-IR] cells) expressing the NMDA R1 subunit (NR1) was determined using double-label immunofluorescence microscopy. An increase in the percentage of TH-IR cells expressing NR1 occurred over time in culture and this correlated with the toxicity of NMDA. At 7 days in vitro (DIV 7), only 17% (n=167 cells/4 experiments) of TH-IR cells expressed NR1 and these cells were insensitive to NMDA toxicity. This increased to 80% (n=254 cells/6 experiments) by DIV 11 and cultures were now susceptible to NMDA-induced injury. Cultures grown for either 7 or 11 days were treated for 48 hr with increasing concentrations of MPP= (0.5-20 microM) and the loss of dopaminergic neurons was determined by cell counting. Cultures at DIV 7 were more sensitive to MPP= than 11-day-old cultures (LD50= approximately 0.75 microM vs. 15 microM, respectively). Co-exposure to MK-801 (5 microM) did not protect against MPP+ toxicity in young cultures, but attenuated MPP+ toxicity in the older cultures, becoming statistically significant at 20 microM MPP+. These data indicate that the activation of NMDA receptors is not required for, but can contribute to, MPP(+)-induced neurodegeneration of dopaminergic cells in culture.

β-Amyloid-Stimulated Microglia Induce Neuron Death via Synergistic Stimulation of Tumor Necrosis Factor α and NMDA Receptors

“…One possibility for this discrepancy could be a result of differences in expression levels of NMDA or TNF␣ receptors. We and others have observed that expression of NMDA and TNF␣ receptors is dependent on culture age, culture density, and the presence of contaminating glia (Daniels and Brown, 2001;Church and Hewett, 2003) (data not shown). Previous culture protocols contained an apparently higher number of glia than ours, and the culture age and neuronal plating density used also differ (Houzen et al, 1997;Furukawa and Mattson, 1998).…”

Preferential vulnerability of mesencephalic dopamine neurons to glutamate transporter dysfunction

“…2003). In this model, the contribution of NMDA‐receptor mediated mechanism increases with increasing MPP + doses (Church and Hewett 2003), and glutamate toxicity through these receptors is exacerbated by low doses of MPP + (Sawada et al. 1996).…”

Characterization of organotypic ventral mesencephalic cultures from embryonic mice and protection against MPP+ toxicity by GDNF

“…In the here‐described mouse VM cultures, the toxicity of MPP + decreased with time in vitro . This is in line with other in vitro studies using primary mesencephalic (Church & Hewett, 2003; Danias et al ., 1989; Koutsilieri et al ., 1995) or sympathetic cell cultures (Bhave et al ., 1996). This may seem in contrast to the in vivo situation, where vulnerability for MPTP increases with ageing (Irwin et al ., 1993; Desai et al ., 1996; Sugama et al ., 2003).…”