Neurons In Spinal Cord Cultures Research Articles

NMDA and non-NMDA receptor-mediated differential Ca2+ load and greater vulnerability of motor neurons in spinal cord cultures

Glutamate receptor activated neuronal cell death has been implicated in the pathogenesis of motor neuron disease but the molecular mechanism responsible for neuronal dysfunction needs to be elucidated. In the present study, we examined the contribution of NMDA and non-NMDA sub-types of glutamate receptors in selective vulnerability of motor neurons. Glutamate receptor activated Ca2+ signaling, mitochondrial functions and neurotoxicity in motor neurons and other spinal neurons were studied in mixed spinal cord primary cultures. Exposure of cells to glutamate receptor agonists glutamate, NMDA and AMPA elevated the intracellular Ca2+, mitochondrial Ca2+ and caused mitochondrial depolarization and cytotoxicity in both motor neurons and other spinal neurons but a striking difference was observed in the magnitude and temporal patterns of the [Ca2+]i responses between the two neuronal cell types. The motor neurons elicited higher Ca2+ load than the other spinal neurons and the [Ca2+]i levels were elevated for a longer duration in motor neurons. AMPA receptor stimulation was more effective than NMDA. Both the NMDA and non-NMDA receptor antagonists APV and NBQX inhibited the Ca2+ entry and decreased the cell death significantly; however, NBQX was more potent than APV. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors contribute to glutamate-mediated motor neuron damage but AMPA receptors play the major role. AMPA receptor-mediated excessive Ca2+ load and differential handling/regulation of Ca2+ buffering by mitochondria in motor neurons could be central in their selective vulnerability to excitotoxicity.

Cultured motor neurons possess calcium-permeable AMPA/kainate receptors.

We examined the biology of AMPA/kainate-induced motor neuron degeneration using dissociated spinal cord cultures and motor neuron-specific antibodies which enable characterization of individual motor neurons in culture. Cobalt, which is thought to pass through Ca2+-permeable AMPA/kainate receptors following kainate exposure, labeled motor neurons in spinal cord cultures. The analysis of AMPA subunit distribution in dissociated motor neurons revealed a unique pattern of glutamate receptor (GluR) subunits in those cells; the GluR1 subunit was found in all spinal cord neurons, but the GluR2 subunit was not found in identified dissociated motor neurons. These data suggest that selective sensitivity of motor neurons to non-NMDA receptor activation is due, at least in part, to the presence of Ca2+-permeable AMPA/kainate receptors.

A chromaffin cell-derived protein induces the NADPH-diaphorase phenotype in cultured rat spinal cord neurons.

We have recently demonstrated that neurotrophins induce reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in cultured spinal cord neurons. One prominent neuron population of the spinal cord expressing NADPH-diaphorase activity in vivo are preganglionic sympathetic neurons, including those innervating the adrenal medulla. These neurons receive trophic support from their target. We have shown previously that chromaffin cells contain as yet unidentified neurotrophic molecules, which may include releasable factors relevant for the survival and differentiation of developing preganglionic sympathetic neurons. We have studied the influence of proteins derived from bovine chromaffin cells and released by nicotine on NADPH-diaphorase expression in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age, NADPH-diaphorase activity becomes apparent in the spinal cord and predominantly expressed in sympathetic nuclei. Similar to brain-derived neurotrophic factor and neurotrophin-4, a heat- and trypsin-sensitive component from chromaffin cells contained in granule preparations up-regulated the number of NADPH-diaphorase-positive neurons in spinal cord cultures. Combined application of this activity and neurotrophin-4 resulted in an additive effect, indicating that the effect of the chromaffin cell-derived active component is not mediated by one of the trk B ligands. This was confirmed by co-treatment studies with the trk-signalling pathway inhibitor K252b, which did not inhibit the effect of the chromaffin cell-derived protein(s). Further studies revealed that NADPH-diaphorase reactivity is inducible in spinal cord neurons at any time point throughout the entire culture period of six days, suggesting de novo induction of the enzyme rather than a survival-promoting effect of the activity from chromaffin cells. Culture supernatants from nicotine-stimulated bovine chromaffin cells induced NADPH-diaphorase-positive neurons at the same magnitude as the material obtained from chromaffin granule preparations. Our data suggest that chromaffin cell-derived proteins are capable of up-regulating NADPH-diaphorase activity or to induce de novo this transmitter phenotype in neuron populations of the spinal cord, which may include preganglionic sympathetic neurons.

The neurotrophins BDNF, NT-3 and -4, but not NGF, TGF-β1 and GDNF, increase the number of NADPH-diaphorase-reactive neurons in rat spinal cord cultures

Neurotrophins have multiple functions for the development of the nervous system. They can promote survival and differentiation of select neuronal populations, but have also been shown to play instructive roles in the determination of the transmitter phenotype of neurons. We have investigated the influence of neurotrophins on the expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), a histochemical marker for nitric oxide synthase, in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age we found NADPH-d reactivity becoming apparent in the spinal cord and predominantly expressed in preganglionic autonomic nuclei. Numbers of NADPH-d-positive neurons in spinal cord cultures were very low 24 h after plating. They did not change significantly until day 4 in vitro. However, treatment with the neurotrophins BDNF, NT-3 or NT-4 significantly increased their numbers. The effect became apparent after just 24 h, and was significant with concentrations as low as 1 ng/ml. Treatment with BDNF, NT-3 and NT-4 also augmented numbers of NADPH-d-positive neurons when initiated after three or five days in culture, and became consistently apparent within 24 h. This suggests that the neurotrophin-mediated increase in NADPH-d-positive neurons is unlikely to be due to promotion of neuron survival. NGF and two members of the transforming growth factor-beta superfamily, which have pronounced trophic effects on select neuron populations in vitro, TGF-beta 1 and GDNF, were not effective. Combined application of NT-4 and NT-3 had no additive effect. Our data therefore suggest that neurotrophins are involved in the developmental regulation of NADPH-d activity in neuron populations of the spinal cord. Neuron populations affected may include preganglionic autonomic neurons. NADPH-d activity may be induced in neurons expressing the enzyme constitutively, yet at undetectable levels, or may be induced de novo.