Distinct Regulation of Bioenergetics and Translation by Gp I mGluR and NMDAR (2020)
Figure 1

Figure 1

mGluR Dependent Protein Synthesis Presents the Bulk of the Metabolic Burden Following Glutamate Stimulation in Cortical Neurons:

(A) Schematic depicting experimental workflow for the measurement of the ATP/ATP+ADP ratio using luciferase-based methods following glutamate stimulation in the presence or absence of various drugs from high-density cultured cortical neurons at 15 days in-vitro (DIV 15).

(B) Bar graph representing the normalized average value of the neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with anisomycin (25μM) and on glutamate stimulation with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. **p<0.01, ***p<0.001, ****p<0.0001, n=5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.

(C) Bar graph representing the normalized average value of neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with MPEP, on glutamate stimulation with CNQX + D-AP5 and on glutamate stimulation with CNQX + D-AP5 + anisomycin. Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment.. *p<0.05, ***p<0.001, n=4-5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.

(D) Representative current-clamp and voltage-clamp traces showing a single neuron firing spontaneous bursts of action potentials. The traces on the right shows a single representative burst at a higher temporal resolution.

(E) Bar graph representing the burst frequency and burst duration both in the voltage and current-clamp mode. n=16 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.

(F) Representative voltage-clamp traces of miniature EPSCs recorded (Vhold= -70mV) from a neuron in presence of TTX.

(G) Bar graph representing the baseline mEPSC frequency and amplitude in the current-clamp mode. N=11 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.

(H) Schematic depicting experimental workflow for the measurement of the synaptic ATP level using luciferase-based methods following DHPG and NMDA treatment in the presence or absence of various drugs from cortical synaptoneurosomes obtained from Sprague Dawley rats at postnatal day 30 (P30).

(I) Immunoblots depicting levels of PSD 95, GFAP, Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations. Quantification representing the average fold enrichments of PSD 95, Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate. Data presented as mean ± SEM with scattered data points. Data points for all the proteins were normalized to their respective whole lysate values. **p<0.05, **p<0.01, n ≥ 7 animals for each group. One-sample t-test.

(J) Bar graph representing the normalized average value of synaptic ATP level on the basal condition, on DHPG treatment for 5 minutes, on DHPG treatment with MPEP, on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. *p<0.05, n=8 animals. One-way ANOVA followed by Bonferroni's multiple comparison test.

(K) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition, on NMDA treatment for 5 minutes and on NMDA treatment with D-AP5. Data presented as mean +/- SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. n=8 independent platings.