146 J
Fig. 2. Age-related glutamate neurotoxicity in cultured neocortical neurons. Photomicrographs from the same fields were taken before left, A and C and after exposure right, B and D to glutamate for 4 h. Magnification 3203. A Control neurons at day 4; B 4-day cells treated with 2 mM glutamate for 4
h; C Control neurons at day 8; D 8-day cells treated with 100 mM glutamate for 4 h. Injured neurons were illustrated by arrowheads. Arrowhead a, swelling. Arrowhead b, disrupted membrane. Arrowhead c, condensed soma. Arrowhead d, irregular cell body. Arrowhead e, disappearance of neurites.
Arrowhead f, disappearance of cells. Note that there was no appreciable injury in 4-day neurons exposed to 2 mM glutamate, whereas more than most of cells were injured in 8-day culture exposed to only 100 mM glutamate, suggesting that glutamate caused an age-dependent injury in neurons.
Radioactivity CPM was counted in a Packard beta statistical analysis with a non-paired, two-tailed Student’s
counter. Non-specific binding was estimated using the t-test. Statistical significance was considered if the P-value
same procedure, with excessive unlabeled DADLE in the was smaller than 0.05.
binding solution.
The specifically-bound
radioactive DADLE was determined by subtracting the non-specific
binding measurement from the total binding using only
3. Results
3
[ H]-DADLE. 3.1. Glutamate-induced neuronal injury is age-dependent
2.8. Statistics Since we [19,55] and others [37] have observed that the
For the morphologic quantification, values obtained neuronal response to stressful conditions depends in a
from two fields in a culture dish were averaged and major way on age, we assessed the effect of glutamate on
considered as one value. All experiments done for the cultured neurons at different days in culture. In all 4–10
morphologic assessment and biochemical assay were per- day cultures, only less than 5 of neurons were found to
formed in at least eight dishes n58 or more from four be injured in sham control same procedures without
different cultures for each group. Receptor binding was glutamate. However, there was a major difference in
studied in six dishes n56 obtained from at least four response to glutamate exposure between 4-day and 8–10
different cultures for each time point. All data are pre- day neurons. When 4-day cultured neurons were exposed
sented as mean values6standard error and subjected to to 0.1–10 mM of glutamate for 4–24 h, no appreciable cell
J . Zhang et al. Brain Research 885 2000 143 –153
147
Fig. 5. LDH activity in cultured neurons after exposure to glutamate at 4 and 8–10 days. LDH activity in neurons was expressed in percent of
control. Results were expressed as the means6S.E.M. n58 for all Fig. 3. Major difference in glutamate-induced neuronal injury at 4 days
groups. w, P,0.001 vs. control. Note that high concentration of versus 8–10 days. Cultured cortical neurons were exposed to glutamate
glutamate up to 2 mM had no apparent effect on LDH levels in 4-day for 4 h. Injury of neurons is expressed as a percentage of pre-treatment.
neurons, but a lower concentration of glutamate 100 mM markedly Data were presented in means6S.E.M. n58 for all groups. w, P,0.001
decreased LDH levels in 8- or 10-day neurons. vs. control no glutamate. Note that there is no increase in neuronal
injury in 4 day cultures after exposure to 0.1–10 mM glutamate, while there was a significant increase in cell injury in 8 and 10 day cultures
exposed to only 100 mM glutamate, suggesting that glutamate-induced
some cells disappearing. As shown in Fig. 4, there was
neuronal injury greatly depends on age.
also a marked increase in LDH level in the medium of 8- or 10-day cultures after glutamate exposure, with a signifi-
cant decrease in intracellular LDH activity Fig. 5. injury ,5 was detected Figs. 2A, B and 3. Also, at
these concentrations of glutamate, LDH level did not significantly change in the culture medium or intracellular-
3.2. d-opioid receptor agonist protects glutamate- ly Figs. 4 and 5. In sharp contrast, glutamate at a
induced neuronal injury concentration as low as 100 mM for 4 h induced significant
cell injury .60 in 8- or 10-day cortical neurons in Since the above experiments demonstrated that gluta-
sister cultures Figs. 2C, D and 3. Injured cells exhibited mate induced neuronal injury in neurons cultured for 8–10
neuronal swelling, neurite blebbing, and, in severe cases, days, we performed the rest of our studies at that age.
neuronal membrane disruption and disintegration with After exposure to 100 mM glutamate for 4 h, more than
65 of the cell population appeared injured n523, P, 0.01; Figs. 6A, B and 7, whereas only about 4 of
neurons were injured if glutamate was not added Fig. 7. A significant increase in medium LDH activity and a
decrease in intracellular LDH activity were also observed when cultures were exposed to the same amount of
glutamate n516, P,0.01, Figs. 8 and 9. Application of DADLE at a concentration of 10 mM simultaneously with
glutamate 100 mM significantly reduced neuronal injury, on average, by almost half under the same test conditions
n521, P,0.05, Figs. 6C, D and 7. We further observed that increasing concentration of DADLE up to 50 mM
could not further reduce neuronal injury n54. With lower concentrations of DADLE 10–100 nM, only a slight
protective effect was evident in some neuronal cultures data not shown. Glutamate-induced LDH increase in the
Fig. 4. LDH levels in culture medium at different ages after glutamate
medium was also significantly reduced by DADLE. As
exposure. LDH activity in the culture medium expressed in percent of
shown in Fig. 8, LDH activity was almost 80 above
control. Results were expressed as means6S.E.M. n58 for all groups.
control level in the glutamate alone group, but only about
w, P,0.001 vs. control. Note that in 4 day cultures exposed to glutamate
30 over control level in the glutamate plus DADLE
up to 2 mM, LDH levels in the medium were similar to those in control,
group n516, P,0.001, Fig. 8. As expected, glutamate-
while in 8 or 10 day cultures, LDH levels were significantly increased in the medium by even 100 mM glutamate.
induced decrease in intracellular LDH activity was re-
148 J
Fig. 6. Protective effect of DADLE against glutamate-induced neuronal injury. Photomicrographs of the same fields were taken before left, A, C and E and after 4 h of cell treatment right, B, D and F in 9-day cultures. Magnification 3203. A and B Glutamate induced injury. A Control
before treatment. B 100 mM glutamate alone. C and D Neuroprotective effect of DADLE. C Control before treatment. D 100 mM glutamate plus 10 mM DADLE. E and F Abolishment of DADLE neuroprotective effect by Naltrindole. E Control before treatment. F 100 mM glutamate
plus 10 mM DADLE and 10 mM Naltrindole. Note that DADLE had a marked protection on glutamate-induced injury and this neuroprotection was completely blocked by Naltrindole at a concentration of 10 mM.
versed by application of DADLE. LDH activity was in these cultured neurons. Receptor binding showed that
significantly higher in neurons exposed to glutamate and d-opioid receptor expression was low at day 4 in culture
DADLE as compared to glutamate only n516, P,0.01, and this increased with age. On day 10, d-opioid receptor
Fig. 9. density in neurons was 3-fold larger in magnitude than at 4
We also determined whether d-opioid receptors existed days P,0.05, n56.
J . Zhang et al. Brain Research 885 2000 143 –153
149
Fig. 9. Effect of d-opioid receptor activation on LDH levels in 8–10 day Fig. 7. Effect of DADLE on glutamate-induced neuronal injury. The
neurons exposed to glutamate. LDH was assayed in the neurons after 4 h population of injured neurons was expressed as a percentage of the
of treatment. Data represent means6S.E.M. n516 for control; n516 for control before treatment. Data represent mean6S.E.M. n513 for
glutamate; n514 for glutamate plus DADLE; n58 for glutamate plus control; n523 for glutamate; n521 for glutamate plus DADLE; n511 for
DADLE and Naltrindole. values obtained from 8 to 16 samples in six glutamate plus DADLE and Naltrindole. w, P,0.01 vs. control. ♦,
cultures. LDH activity in neurons was expressed in percent of the control. P,0.01 vs. glutamate plus DADLE. Note that DADLE 10 mM had a
w, P,0.01 vs. control. ♦, P,0.05 vs. group of glutamate plus DADLE. significant neuroprotective effect on glutamate-induced neuronal injury
j, P,0.01 vs. group of glutamate plus DADLE. Note that LDH levels in and the protective effect of DADLE was completely blocked by 10 mM
neurons are decreased with 100 mM glutamate and this decrease is Naltrindole.
reversed by 10 mM DADLE. Also note that DADLE effect is completely blocked by 10 mM Naltrindole.
3.3. d-receptor antagonist completely abolishes the neuroprotective effect by DADLE
To verify whether the protective effect of DADLE is mediated by d-opioid receptors, a d-opioid antagonist,
Naltrindole 10 mM, was added simultaneously with glutamate 100 mM and DADLE 10 mM. Microscopic
analysis revealed that administering Naltrindole reversed the neuroprotective effect of DADLE. In neuronal cultures
exposed to glutamate plus DADLE, as shown in Fig. 7, glutamate-injured neurons were reduced by almost half
with existence of DADLE, while adding Naltrindole completely abolished this neuroprotection at the same time
in sister cultures. In some cases, neuronal injury was even worse in Naltrindole-treated culture than glutamate only
culture. On average, about 67 of neurons were injured in this group of glutamate plus DADLE and Naltrindole
n511, P,0.01, Figs. 6E, F and 7. This is similar to that of glutamate only group Fig. 7.
Measurements of LDH release showed similar results. In
Fig. 8. Inhibition of glutamate-induced LDH release from neurons into
culture media with glutamate only, LDH activity was
media by activation of d-opioid receptors in 8–10 day cultures. LDH was
79.4611.8 higher than control n516, P,0.01; Fig. 8.
measured in culture medium after 4-h of cell treatment. Data represent means6S.E.M. values n516 for control; n516 for glutamate; n516 for
This increased LDH level was markedly attenuated by
glutamate plus DADLE; n58 for glutamate plus DADLE and Naltrin-
DADLE. Fig. 8 shows that the media containing both
dole. LDH activity in culture medium was expressed in percent of the
glutamate and DADLE had an LDH level of 33.166.7
control. w, P,0.01 vs. control. ♦, P,0.05 vs. glutamate plus DADLE.
higher than control. As compared to glutamate only group,
j, P,0.01 vs. glutamate plus DADLE. Note that LDH is increased with
the increased LDH level was reduced by 58.3 n516,
100 mM glutamate and this increase is reduced by 10 mM DADLE. DADLE effect is completely abolished by 10 mM Naltrindole.
P,0.01. Cells exposed to glutamate as well as DADLE
150 J
and Naltrindole showed a significantly higher activity level in the media, i.e., 192.568.8 of control n58, P,0.01
when compared to the group of glutamate and DADLE; Fig. 8.
Intracellular LDH activity after glutamate exposure was significantly reduced by more than 20 78.763.4 in the
glutamate group vs. 100 in control, n516, P,0.01; Fig. 9. This decrease could be largely reversed by DADLE
n514, P,0.01 as shown in Fig. 9. Upon administration of Naltrindole, the effect of DADLE on LDH activity in
neurons was completely abolished and reached a level of 78.46 2.9 of control n58 which was very similar to
that of glutamate alone group P.0.05, Fig. 9.
3.4. Activation of m- or k-opioid receptors has no appreciable effect on glutamate-induced neuronal injury
To determine whether m- or k-opioid receptors have an effect on glutamate-induced injury, we used DAMGO and
U50488H to stimulate m- and k-opioid receptors respec- tively in exactly the same experimental conditions as in the
studies of d-opioid receptors. LDH measurements showed that 100 mM glutamate induced a similar change in LDH
release as shown above, i.e., a marked increase in LDH level in the culture medium 122.1619.3 over control,
n58, P,0.01. This increase in LDH level was not affected by 5 mM of DAMGO or U50488H. In the culture
groups of glutamate plus DAMGO or U50488H, LDH activity increased by 133.4635.8 and 141.6633.1
respectively. There was no statistical difference between these groups and the glutamate only group n58, P.0.05.
Adding m- or k-opioid receptor antagonists b-FNA or nor-BNI, 10 mM to opioid agonist groups had no effect on
Fig. 10. Effects of m- and k-opioid receptor activation and inhibition on
LDH levels in the media n58, P.0.05. To determine
glutamate-induced LDH release from neurons into media. Experiments
whether an increase in m- or k-opioid agonist concen-
were performed in the same way as in Fig. 8 except for application of
trations induces neuroprotection, we repeated the above
DAMGO, b-FNA panel A, U50488H and nor-BNI panel B. Data
experiments with 10 mM DAMGO and U50488H and
represent means6S.E.M. values. LDH activity in culture medium was
found similar results Fig. 10.
expressed in percent of the control. w, P,0.01 as compared to control n58 for all groups. Note that m- or k-opioid agonists and antagonists
The measurement of intracellular LDH activity further
had no appreciable effect on glutamate-induced neuronal injury.
demonstrated that m- or k-opioid receptors have no neuro- protection against glutamate-induced injury. In the group
of glutamate alone, LDH level in neurons decreased by ceptors, we limited our analysis to LDH measurement and
21.864.7 as compared to control level n57, P,0.01. did not assess the neuronal injury with more time-consum-
In glutamate plus 5 mM DAMGO group from sister ing morphological quantification.
cultures, LDH level in neurons showed a decrease by 28.266.3 as compared to control level n57, P,0.01
when compared to control group, P.0.05 when compared
4. Discussion
