Opposing effects of excitatory amino acids on chick embryo spinal cord motoneurons: excitotoxic degeneration or prevention of programmed cell death

Abstract

Acute administration of a single dose of NMDA on embryonic day (E) 7 or later induces a marked excitotoxic injury in the chick spinal cord, including massive necrotic motoneuron (MN) death. When the same treatment was performed before E7, little, if any, excitotoxic response was observed. Chronic treatment with NMDA starting on E5 prevents the excitotoxic response produced by a later "acute" administration of NMDA. Additionally, chronic NMDA treatment also prevents the later excitotoxic injury induced by non-NMDA glutamate receptor agonists, such as kainate or AMPA. Chronic NMDA treatment also reduces normal MN death when treatment is maintained during the period of naturally occurring programmed cell death (PCD) of MNs and rescues MNs from PCD induced by early peripheral target deprivation. The trophic action of chronic NMDA treatment appears to involve a downregulation of glutamate receptors as shown by both a reduction in the obligatory NR1 subunit protein of the NMDA receptor and a decrease in the kainate-induced Co(2+) uptake in MNs. Both tolerance to excitotoxicity and trophic effects of chronic NMDA treatment are prevented by the NMDA receptor antagonist MK-801. Additionally, administration of MK-801 alone results in an increase in MN PCD. These data indicate for the first time that early activation of NMDA receptors in developing avian MNs in vivo has a trophic, survival-promoting effect, inhibiting PCD by a target-independent mechanism that involves NMDA receptor downregulation.

Fig. 1.

Nissl-stained sections from E10 chick embryo lumbar spinal cords after different regimens of treatment. Top rows, Low-magnification views of a hemisection of a spinal cord, with the LMC indicated in dots.Bottom rows, High magnification of the corresponding LMC. A, B, and C were treated with saline from E5 to E9, whereas A′,B′, and C′ received the standard trophic NMDA treatment. At the end of the treatment, embryos received a pulse of saline (A, A′), 0.25 mg NMDA (B, B′), or 0.5 mg kainate (C, C′) on E10 and were killed 12 hr later. Note that NMDA trophic treatment results in an enlargement of the LMC and protects against acute damage induced by pulses of excitotoxins. Scale bar (in A): top rows, 200 μm; bottom rows, 50 μm.

Fig. 2.

Number of surviving MNs (A) and pyknotic cells (B) in the lumbar LMC in saline (control) or trophic NMDA-treated embryos. A, Note that the excessive number of rescued MNs present on E9–E10 after trophic NMDA treatment is progressively lost in the subsequent developmental period. B, The peak of pyknotic profiles normally present on E8 in control embryos is significantly reduced by trophic NMDA treatment. Subsequently, there is an increase in the number of pyknotic cells in NMDA-treated embryos coincident with the loss of NMDA-rescued MNs. Each point represents the mean ± SEM of four to eight embryos. Error bars are sometimes smaller than symbols. *p < 0.05, **p < 0.01 versus saline (Student's t test).

Fig. 3.

Effects of either saline or trophic NMDA treatment on the number of active embryonic movements. The developmental pattern of motor activity is drastically changed by trophic NMDA treatment. The high values of error bars on E9 and E10 NMDA-treated embryos are caused by large variations in movements commonly observed at these ages as a consequence of NMDA treatment. Each point represents the mean ± SEM of four to eight embryos. Error bars are sometimes smaller than symbols. *p < 0.05, **p < 0.01 versus saline (Student's t test).

Fig. 4.

Effect of trophic NMDA treatment on MN survival after LBR performed on E2. A, B, Photomicrographs taken from E9 LBR operated embryos after treatment from E5 to E8 with either saline (A) or NMDA (B). Note the dramatic depletion of MNs in the LMC on the operated side of saline-treated embryos (arrow in A) and compare with the increased number of MNs in NMDA-treated embryos on the operated side (arrow in B). Scale bar (inA): A, B, 200 μm.C, Number (mean ± SEM) of MNs in lumbar LMC of embryos from LBR experiments. *p < 0.01 versus saline nonoperated; **p < 0.01 versus saline operated (Student's t test). Numbers in parentheses indicate sample sizes.

Fig. 5.

Identification of NR1 protein in Western blots of spinal cord membrane fractions from E10, E11, E12, and E16 embryos.S, Saline; N, after chronic NMDA treatment. The ratio (mean ± SEM) of densitometric values from saline versus NMDA treatment is plotted for each experimental condition. Numbers in parentheses in bars indicate sample sizes; each sample represents the total membrane fraction obtained from at least 20 spinal cords. Legends inx-axis indicate the embryonic day of sampling and the duration of treatment (in parentheses). Note that, when NMDA treatment is stopped, the downregulation of NR1 protein is reversed.

Fig. 6.

Co²⁺ uptake in lumbar LMC MNs of E10 (A, B, E) and E16 (C, D, F) chick embryos after 15 min stimulation with 100 μm kainate. Embryos were treated previously from E5 to E9 with either saline (A, C, E,F) or the trophic protocol of NMDA (B, D). Spinal cord preparations were also stimulated in the presence of CNQX (30 μm;E, F). Sections were counterstained with thionin. Note that the trophic treatment with NMDA greatly reduces Co²⁺ uptake in MNs on E10 (A, B) but not on E16 (C,D) embryos. Kainate stimulation in the presence of CNQX abolishes Co²⁺ uptake (E,F). Scale bar (in E):A, B, E, 50 μm;C, D, F, 100 μm.

Fig. 7.

Morphometric evaluation of kainate-induced Co²⁺ uptake. Graphs show the percentage (mean ± SEM) of the Co²⁺-labeled area in the LMC of spinal cord of E10 (A) and E16 (B) chick embryos treated with either saline or NMDA from E5 to E9. *p < 0.001 versus saline (Student's t test). Numbers in parentheses indicate sample sizes.

Fig. 8.

MK-801 increases motoneuron death during the period of normal PCD. Number (mean ± SEM) of pyknotic cells in the lumbar LMC of chick embryos after treatment with either saline or different doses of MK-801 on E7. Counts were made 12 hr after the treatment. Numbers in bars indicate the sample size. *p < 0.01, Student's t test.