Effects of NMDA and its antagonists on ventral horn cholinergic neurons in organotypic roller tube spinal cord cultures

Abstract

Neurotoxic effects of excitatory amino acid (EAA) receptor agonist N-methyl-D-aspartic acid (NMDA) and its antagonists on ventral horn cholinergic neurons were studied in organotypic rollertube cultures of spinal cord (OTC-SCs) using biochemical assays of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and AChE histochemistry. NMDA exposure decreased ChAT and AChE activity by 83% and 66%, respectively. Cultures treated with NMDA also showed a marked loss of AChE staining in both dorsal and ventral horns and a significant, dose-dependent decrease in the number of ventral horn AChE-positive neurons (VHANs). NMDA treatment primarily resulted in the loss of small VHANs (< 300 microns2). VHANs with a size and distribution typical of alpha-motoneurons were relatively well preserved. The effects of NMDA on OTC-SCs appeared to be independent of the age of the cultures. The NMDA antagonist DL-AP5 completely prevented the NMDA-induced loss of ChAT activity, but only attenuated the effect of NMDA on ChE activity. The antagonists DL-AP5, D-AP5 and MK-801, used alone, caused significant loss and/or shrinkage of VHANs. These effects appeared to be distinct from the NMDA mediated toxicity. The results indicate that NMDA and its antagonists exert powerful toxic effects on ventral horn cholinergic neurons. The large cholinergic alpha-motoneurons, however, appear to be relatively immune to these toxic effects.

PGE2 receptors rescue motor neurons in a model of amyotrophic lateral sclerosis

“…In the organotypic spinal cord model, chronic application of glutamate transporter inhibitors results in elevated extrasynaptic glutamate levels and a gradual loss of motor neurons over several weeks to months 18, 22, 24, 35. This model has been used extensively to examine mechanisms of neurodegeneration36–38 and to evaluate potential therapeutic strategies, including growth factor administration 19, 22–24, 35…”

Ca2+ entry pathways in mouse spinal motor neurons in culture following in vitro exposure to methylmercury

“…There are numerous reports correlating activation of EAA receptor channels with Ca 2+ -mediated excitotoxicity in spinal motor neurons (Delfs et al, 1997; Feldman et al, 1997; Urushitani et al, 2001). To test whether glutamate receptor-mediated pathways contributed to the MeHg-induced increase in [Ca 2+ ] i , we exposed cells to various EAA inhibitors prior to MeHg in an attempt to block or delay the MeHg-induced increase in fura-2 fluorescence ratio.…”

“…The most likely source is endogenous glutamate released into the medium in the presence of MeHg, a potent action demonstrated in isolated brain slices (Yuan and Atchison, 2007). This glutamate could then auto-activate adjacent motor neurons (Schramm et al, 1990; Delfs et al,1997; Feldman et al, 1997; Urushitani et al, 2001). However, spinal motor neurons are cholinergic, not glutamatergic, so other cell types in the mixed culture must have been the source of this putative glutamate.…”

Calcium mediated excitotoxicity in neurofilament aggregate-bearing neurons in vitro is NMDA receptor dependant

“…Although an exact mechanism for motor neuron degeneration in ALS has not yet been elucidated, both abnormal neurofilament (NF) metabolism [2][3][4] and excitotoxicity [5][6][7][8][9] are considered to be key components of the disease process.…”