Synaptic and Nuclear Signaling Are Differentially Regulated by Activity During Neuronal

Supplementary Figure 1 NMDA stimulation causes LDH release from damaged cells in a dose- dependent manner. DIV 14 cortical neurons were treated with 0, 15, 20, 30, 40, and 50 uM

NMDA for 30 min. Twenty four hours after NMDA withdrawal, LDH level in the culture medium and neurons were separately determined by using the Cytotoxicity Detection Kit from

Roche Applied Sciences. The relative LDH level was calculated as (LDH in medium)/(LDH in medium plus LDH in neurons), and arbitrarily designated as 1 for the control group. One-way

ANOVA and the post hoc Student–Newman–Keuls analysis revealed significant difference (p <

0.05) among different groups with a < b < c < d.

Supplementary Figure 2 Selective blockage of syn-NMDAR. (A) and (B) MK801 irreversibly blocks the opened synaptic receptors. (A) Calcium imaging traces from individual neurons treated with bicuculline/MK801 (as indicated by the line for the duration of application). (B)

Bicuculline (as indicated by the line for the duration of application) fails to induce significant calcium influx in neurons pre-treated with bicuculline/MK801. DIV 14 cortical neurons were first co-treated with bicuculline and MK801 for 2 min, and then washed extensively. Calcium imaging was used to determine calcium influx upon bicuculline application. (C) MK801 blocks bicuculline-induced CREB phosphorylation. Neurons at DIV 14 were treated with bicuculline or bicuculline plus MK801 for 30 min. Samples were analyzed for the level of p-CREB by Western blot. Representative blot is shown in the upper panel, and quantification is shown in the lower panel. (D) Bicuculline fails to activate CREB in neurons pre-treated with bicuculline/MK801.

DIV 14 neurons were first pre-treated with bicuculline plus MK801 for 2 min, washed, and then subjected to a 30-min incubation with bicuculline. The level of p-CREB was determined by

Western blot. NS: not significant.

1 Supplementary Figure 3 Blocking synaptic receptors results in resistance to NMDAR- dependent excitotoxicity. DIV 14 neurons were first pre-treated with bicuculline and MK801 for

2 min followed by extensive wash with conditioned medium. Subsequently, neurons were incubated with NMDA or glutamate (concentrations as indicated) for 30 min (A) or 24 hours (B).

Neurons were fixed 20 hours (A) or immediately (B) after the treatment, and stained with DAPI.

Cell death was determined by the appearance of condensed nuclear staining. *: p < 0.05 when compared to other groups. Note that 100 uM glutamate triggered significant death in neurons not pre-treated with bicuculline/MK801 (A).

Supplementary Figure 4 Brief activation of synaptic receptor does not block NMDAR- dependent excitotoxicity. DIV 14 neurons were first pre-treated with bicuculline or vehicle for 2 min followed by extensive wash with conditioned medium. Subsequently, neurons were incubated with 50 M NMDA for 30 min. Neurons were fixed 20 hours later, and stained with

DAPI. Cell death was determined by the appearance of condensed nuclear staining. NS: not statistically significant.

Supplementary Figure 5 NMDAR-independent cell death during OGD. DIV 14 cortical neurons were first treated with bicuculline plus MK801 for 2 min, and subjected to OGD for 2 hours. The neurons were then incubated with conditioned culture medium for 20 hours, and the degree of cell death was determined by DAPI staining.

2 Supplementary Figure 6 Activation of Ex-NMDAR does not cause significant LDH release.

DIV 14 cortical neurons were first treated with bicuculline and MK801 for 2 min, and then incubated with different concentrations of NMDA (as indicated) for 30 min. The relative LDH level was determined 24 hours after the withdrawal of NMDA as describe in Supplementary

Figure 1.

Supplementary Figure 7 Activation of receptors that are only sensitive to high concentration

NMDA does not cause significant LDH release. DIV 14 cortical neurons were first treated with

15 uM NMDA and MK801 for 3 min, and then incubated with different concentrations of

NMDA (at 0, 15, and 50 uM) for 30 min. The relative LDH level was determined 24 hours after the withdrawal of NMDA as describe in Supplementary Figure 1.

Supplementary Figure 8 Extended duration of NMDA stimulation deactivates CREB in a dose- dependent manner. DIV 14 neurons were treated with 15 (A) or 20 uM NMDA (B) for 0, 30, or

60 min. The level of CREB phosphorylation was determined by Western blots, and normalized to the level of beta-actin.

Supplementary Figure 9 Extended duration of mild NMDA stimulation causes significant cell death. DIV 14 neurons were treated with 20 uM NMDA for 24 hours. Cells were fixed immediately after stimulation, and cell death was determined by DAPI staining.

Supplementary Table 1 List of the up-regulated and down-regulated genes triggered by the activation of syn-NMDAR.

3 Supplementary Table 2 List of the up-regulated and down-regulated genes triggered by the activation of ex-NMDAR.