Fig. 5

mTORC1 acts in combination with synaptically derived signals to enhance spine head diameter. (a-b) Mean (+/−SEM) normalized spine head width and cumulative probability distribution and of cells treated with a subthreshold (10 μM) or super-threshold (400 μM) concentration of glycine assessed 15 min (a) and 45 min (b) post-stimulation. *p < 0.05 relative to vehicle treated controls. Spines treated with a sub-threshold dose of glycine do not exhibit significant changes in head width compared to controls at either time point assessed. (c) Timecourse of head expansion in neurons expressing eGFP alone or in combination with genetic upregulation of mTORC1 activity via RhebQ64L expression after sub-threshold (10 μM) glycine treatment. Co-occurrence of increased mTORC1 signaling with synaptic activation elicited a rapid increase in spine head diameter observed 15 m post stimulation. (d) Cumulative probability distribution and mean +/−SEM values (inset) of altered spine head diameter at 15 m post stimulation shown as percentage of baseline values. HBS alone, n = 255 across 3 cells; 10 μM glycine, n = 595 across 8 cells; RhebQ64L alone, n = 1227 across 7 cells; RhebQ64L + 10 μM glycine, n = 1076 across 9 cells. (e-g) Scatter plots comparing raw spine head values pre vs 15 m post experiment initiation under conditions of sub-threshold glycine stimulation (e), expression of RhebQ64L alone (f), or both stimuli combined (g). Bar graphs on right hand side represent spines that increase (black), decrease (white) or remain stable (gray) as a proportion of total spines in each condition. Spines subject to paired activation RhebQ64L expression with sub-threshold glycine application exhibits increases in head growth well above either condition alone. (h) Mean (+/− SEM) of altered head diameter 15 min post stim, represented a percent change from baseline values separated according to morphological category as indicated. Rapid spine expansion after 15 m under conditions of paired synaptic stimulation and mTORC1 activation are specific to mushroom spines (10 μM glycine mushroom n = 185; RhebQ64L alone mushroom n = 336; RhebQ64L + 10 μM glycine mushroom n = 260), thin spines (10 μM glycine thin n = 57; RhebQ64L alone thin n = 169; RhebQ64L + 10 μM glycine thin n = 185), and filopodia (10 μM glycine fil n = 55; RhebQ64L alone fil n = 90; RhebQ64L + 10 μM glycine fil n = 53). Stubby (10 μM glycine stubby n = 186; RhebQ64L alone stubby n = 395; RhebQ64L + 10 μM glycine stubby n = 362) and flat spines (10 μM glycine flat n = 122; RhebQ64L alone flat n = 247; RhebQ64L + 10 μM glycine flat n = 226) show no significant differences across experimental groups