Chronic Activation of Gp1 mGluRs Leads to Distinct Refinement of Neural Network Activity through Non-Canonical p53 and Akt Signaling

Burst activity is at the baseline after chronic activation of Gp1 mGluRs. Representative traces of burst activity from WT cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14. Traces are from the same designated electrode “before” and “after” drug treatments. Quantification of burst duration and relative number of spikes per burst by comparing “after treatment” to “before treatment” during the 15-min recordings from the same cultures is on the right; n = 7 and n = 6 independent cultures for DMSO and DHPG treatment groups, respectively. Student’s t test was used. Data are represented as mean ± SEM with ns: non-significant.

Chronic activation of Gp1 mGluRs leads to proteasomal degradation of Akt. A, Representative Western blottings of Akt, p-Akt, ERK, p-ERK, and Gapdh, and their quantification from WT cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14 (n = 8 and n = 9 sets of cultures for detecting Akt and ERK, respectively). B, RT-qPCR of Akt mRNA, using two different pairs of primers, normalized to Actin mRNA from WT cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14 (n = 4). C, Representative Western blottings of Ubiquitin and Akt after IP with anti-Akt antibody using lysates from WT cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14 (n = 4). D, Representative Western blottings of Akt, ERK, and Gapdh and the quantification from WT cortical neuron cultures treated with DMSO, DHPG (100 μM), MG132 (10 μM), and DHPG+MG132 at DIV14 (n = 4). MG132 was applied during the second 12 h of DMSO or DHPG treatments. For the quantification above, Student’s t test (A–C) or a two-way ANOVA with Tukey’s test (D) were used. Data are represented as mean ± SEM with *p < 0.05, **p < 0.01, ns: non-significant.

Chronic activation of Gp1 mGluRs reduces Mdm2-p53 signaling. A, Representative Western blottings of Mdm2, and Gapdh from WT cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14 (n = 4). B, Representative Western blottins of p53, and Gapdh from WT cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14 (n = 8). C, Representative Western blottings of Ubiquitin and p53 after IP with anti-p53 antibody using lysates from WT cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14 (n = 3). Student’s t test was used. Data are represented as mean ± SEM with *p < 0.05, **p < 0.01.

p53 is required for chronic activation of Gp1 mGluRs-induced downregulation of Akt. A, Quantification of p53 expression and representative Western blottings of p53 and Gapdh from p53^{f/+-Emx1-Cre+} or p53^{f/+-Emx1-Cre+;} cortical neuron cultures at DIV14 (n = 3). B, Quantification of Akt expression and representative Western blottings of p53 and Gapdh from p53^{f/+-Emx1-Cre+} or p53^{f/+-Emx1-Cre+} cortical neuron cultures at DIV14 (n = 3). C, Quantification of Akt expression and representative Western blottings of Akt and Gapdh from p53^{f/+-Emx1-Cre+} or p53^{f/+-Emx1-Cre+} cortical neuron cultures treated with DMSO or DHPG (100 μM) for 24 h at DIV14 (n = 7 and n = 9 for p53 p53^{f/+-Emx1-Cre+} or p53^{f/+-Emx1-Cre+} cultures, respectively). Student’s t test was used. Data are represented as mean ± SEM with *p < 0.05, **p < 0.01, ns: non-significant.

Chronic activation of Gp1 mGluRs elicits effects on neural network activity in p53^{f/+-Emx1-Cre-} cortical neuron cultures similar to those in WT (C57BL/6J) cortical neuron cultures. A, B, Quantification of relative spontaneous spike frequency (A, left), synchrony (A, right), burst duration (B, left) and relative number of spikes per burst (B, right) from p53^{f/+-Emx1-Cre-} cortical neuron cultures treated with vehicle (DMSO) or DHPG (100 μM) for 24 h at DIV14. The analysis was done by comparing “after treatment” to “before treatment” of the same cultures during the 15-min recordings. Student’s t test was used. Data are represented as mean ± SEM with *p < 0.05, ns: non-significant.

Pharmacological inhibition of Akt partially restores spontaneous network activity in p53^{f/+-Emx1-Cre+} cortical neuron cultures. A, Raster plots of spontaneous spikes from representative 1-min recordings of p53^{f/+-Emx1-Cre+} cortical neuron cultures treated with DMSO, DHPG (100 μM), DMSO+MK-2206 (1 μM), or DHPG+MK-2206 at DIV14. Quantification of relative spontaneous spike frequency and synchrony index by comparing “after treatment” to “before treatment” of the same cultures during the 15-min recordings (n = 5–7 independent cultures). B, Representative average traces of spike amplitude from 1-min recordings of WT cortical neuron cultures treated with DMSO, DHPG (100 μM), DMSO+MK-2206 (1 μM), or DHPG+MK-2206 at DIV14. In the traces, the black lines represent the average of all the spikes within representative 1-min recordings. Traces are from the same designated electrodes before and after treatments (n = 5–7 independent cultures). Quantification of spike amplitude is done by comparing “after treatment” to “before treatment” during the 15-min recordings from the same cultures. For both A, B, MK-2206 was applied during the last 1 h of 24-h DHPG or DMSO treatments. A two-way ANOVA with Tukey’s test was used. Data are represented as mean ± SEM with *p < 0.05, ns: non-significant.

Aberrant burst activity in p53^{f/+-Emx1-Cre+} cortical neuron cultures is normalized by pharmacological inhibition of Akt. Representative traces of burst activity from p53^{f/+-Emx1-Cre+} cortical neuron cultures treated with DMSO, DHPG (100 μM), DMSO+MK-2206 (1 μM), or DHPG+MK-2206 at DIV14. MK-2206 was applied during the last 1 h of 24-h DHPG or DMSO treatments. Traces are from the same designated electrode “before” and “after” drug treatments. Quantification of burst duration and relative number of spikes per burst by comparing “after treatment” to “before treatment” during the 15-min recordings from the same cultures was on the right (n = 6 independent cultures). A two-way ANOVA with Tukey’s test was used. Data are represented as mean ± SEM with *p < 0.05, ns: non-significant.