Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity

Abstract

We have developed "pure" neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2-4 d in vitro, deprived of glial-conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial ("mixed") cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N-methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity.

The Neuroprotective Activity of Group-II Metabotropic Glutamate Receptors Requires New Protein Synthesis and Involves a Glial–Neuronal Signaling

“…In addition, mGlu3 receptors are found in glial cells (Tanabe et al, 1993;Petralia et al, 1996), whereas evidence for the presence of group-III mGlu receptors in astrocytes is lacking. A role for astrocytes in neuroprotection was suggested by the evidence that cortical neurons in pure cultures are more sensitive to toxicity induced by acute exposure to glutamate, by 24 hr exposure to NMDA, or by oxygen-glucose deprivation (Dugan et al, 1995), and less sensitive to the protective activity of DCG-IV against kainate-induced degeneration (our unpublished observation) than neurons in mixed cultures. We have hypothesized therefore either that astrocytes produce a permissive factor that Figure 5.…”

“…This defensive mechanism, however, may not be efficient against all types of excitotoxic insults, because activation of group-II mGlu receptors does not protect cortical neurons grown in mixed cultures against AMPA toxicity Buisson and Choi, 1995;Buisson et al, 1996), whereas the effect on kainate-induced toxicity is still controversial. It is noteworthy that the presence of astrocytes exacerbates AMPA neurotoxicity, although it protects cultured neurons against glutamate or NMDA toxicity (Dugan et al, 1995).…”

Co-administration of Ciliary Neurotrophic Factor with Its Soluble Receptor Protects against Neuronal Death and Enhances Neurite Outgrowth

“…The survival of neurons placed in primary culture is compromised in the absence of growth factors or glial cells, with the majority of cells dying within the first 3-5 days (27,37). Given the broad effects of Complex to promote cell survival, we tested the ability of the agent to promote or enhance the survival of neuron-enriched cultures.…”

“…Neurons maintained in culture for two different periods of time were employed to assess the effects of cytokine treatment on developing neurons and on neurons of a more mature state (27). At no time point examined did CNTFR␣ significantly alter LDH release, compared with vehicle treatment, in either the 3 DIV (Fig.…”

“…Neurons, obtained from the neocortices of embryonic CD-1 mice aged 16 days gestation, were then seeded on top of the astrocytes and allowed to settle for 2 h before replacing the medium with Medium I (54 ml of Neurobasal Medium, 36 ml of Dulbecco's modified Eagle's medium/F-12, 1 ml of B-27 supplements; Invitrogen). On day 4, 2 M cytosine arabinoside was added during a fresh medium change for 24 h. Thereafter, two-thirds of the conditioned medium was replaced every 3 days with fresh Medium I. Co-cultures were maintained for 2 weeks prior to experiments to ensure expression of functional glutamate receptors by the neurons (27).…”