AMPAergic blockade reduces burst frequency and overall spike rate. A) Network bursts can be identified by detected spikes (red dots) time-locked in multiple channels of the MEA (Y axis). One burst (highlighted in red rectangle) is expanded in time and shown in the raster plot on the right. This is illustrated before CNQX (top) and then repeated below at 2hrs, 10 hrs, and 24 hrs following the addition of CNQX. B) The normalized burst rate is shown in control cultures and following application of CNQX for 24 hrs. C) The normalized overall spike rate is shown in control cultures and following CNQX addition over 24 hrs. The mean differences at different time points are compared to control and displayed in Cumming estimation plots. Significant differences denoted by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Recordings from single cultures (filled circles), where mean values (represented by the gap in the vertical bar) and SD (vertical bars) are plotted on the upper panels. Mean differences between control and treated groups are plotted on the bottom panel, as a bootstrap sampling distribution (mean difference is represented by a filled circles and the 95% CIs are depicted by vertical error bars).

Both activity and AMPAR blockade cause a reduction in mlPSC amplitudes that appear to scale down. A) CNQX and TTX produce a reduction in average amplitude of mlPSCs as shown in the scatter plot. The mean differences are compared to control and displayed in Cumming estimation plots. Significant differences denoted by *** p ≤ 0.001. GABAergic mPSC amplitudes from single neurons (filled circles), where mean values (represented by the gap in the vertical bar) and SD (vertical bars) are plotted on the panels to the left. Mean differences between control and treated groups are plotted on the panel to the right, as a bootstrap sampling distribution (mean difference is represented by a filled circles and the 95% Cls are depicted by vertical error bars). Example traces showing mlPSCs are shown below. B) Scaling ratio plots show the relationship of mlPSC amplitudes from treated cultures compared to untreated cultures. All recordings taken from cultured neurons plated on coverslips, not MEAs.

MEA recordings show that optogenetic stimulation restores spiking activity in cultures treated with CNQX. A) Spontaneously-occuring bursts of spiking are identified (synchronous spikes/red dots). Expanded version of raster plot highlighting 2 bursts is shown below. B) Same as in A, but after CNQX was added to the bath and bursts were now triggered by optogenetic stimulation (blue line shows duration of optogenetic stimulation). C-D) Average burst rate (C) or spike rate (D) is compared for CNQX-treated cultures with optogenetic stimulation and control unstimulated cultures at 1hr, 3hrs, 6hrs, and 24hrs after addition of CNQX or vehicle (same control data presented in Figure 4). The mean differences at different time points are compared to control and displayed in Cumming estimation plots. Significant differences denoted by * p ≤ 0.05, *** p ≤ 0.001. Recordings from single cultures (filled circles), where mean values (represented by the gap in the vertical bar) and SD (vertical bars) are plotted on the upper panels. Mean differences between control and treated groups are plotted on the bottom panel, as a bootstrap sampling distribution (mean 27 difference is represented by a filled circles and the 95% Cls are depicted by vertical error bars).

Optogenetic restoration of spiking activity in the presence of AMPAR blockade prevents GABAergic downscaling observed in CNQX alone. A) Scatter plot shows AMPAR blockade triggers a reduction in mlPSC amplitude compared to controls that is prevented when combined with optogenetic stimulation (optostim). The mean differences are compared to control and displayed in Cumming estimation plots. Significant differences denoted by ** p ≤ 0.01, *** p ≤ 0.001. GABAergic mPSC amplitudes from single neurons (filled circles), where mean values (represented by the gap in the vertical bar) and SD (vertical bars) are plotted on the upper panels. Mean differences between control and treated groups are plotted on the bottom panel, as a bootstrap sampling distribution (mean difference is represented by a filled circles and the 95% Cls are depicted by vertical error bars). B) Scaling ratio plots show largely multiplicative relationships to control values for both CNQX and CNQX + photostimulation treatments. Cultured neurons for these recordings were obtained from cells plated on MEAs (control, CNQX, and CNQX+optostim).

GABAergic upscaling was also triggered by changes in spiking activity rather than AMPAR activation. MEA recordigns show that CTZ trended toward increases of both burst rate (A) and overall spike frequency (B) during the 24 hr application, and achieved significance at the 3 hr timepoint for spike frequency. C) CTZ treatment (dissolved in 1:1000 dilution of ethanol (EtOH)) led to an increase in mIPSC amplitude compared to control cultures (equivalent volume of 1:1000 ethanol solution). D) CTZ combined with TTX (in 1:1000 ethanol) produced a reduction of mIPSC amplitude compared to controls (that was no different than TTX alone). The mean differences at different time points or conditions are compared to control and displayed in Cumming estimation plots. Significant differences denoted by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Recordings from single cultures (filled circles), where mean values (represented by the gap in the vertical bar) and SD (vertical bars) are plotted on the upper panels. Mean differences between control and treated groups are plotted on the bottom panel, as a bootstrap sampling distribution (mean difference is represented by a filled circles and the 95% CIs are depicted by vertical error bars). E) Scaling ratios show that both CTZ-induced increases and CTZ+TTX-induced decreases were multiplicative. All mIPSC amplitudes recorded from cultures plated on coverslips, not MEAs.

GABAergic upscaling is triggered by increased spiking activity rather than reduced GABAR activation. Bicuculline-treated cultures (24hrs) plated on MEA’s trended upward in burst frequency (A) and overall spike frequency (B). Recordings from single cultures (filled circles), where mean values (represented by the gap in the vertical bar) and SD (vertical bars) are plotted on the upper panels. C) Bicuculline treatment (24hrs) produced an increase in mlPSC amplitudes. The mean difference is compared to control and displayed in Cumming estimation plots. Significant difference denoted by * p :5 0.05. Recordings from single neurons (filled circles), and mean values (represented by the horizontal line). Control and treated group is plotted, as a bootstrap sampling distribution (mean difference is represented by a filled circles and the 95% Cl is depicted by vertical error bar). D) Ratio plots for bicuculline-induced increase in mlPSCs exhibits a multiplicative profile. All mlPSC amplitudes recorded from cultures plated on coverslips, not MEAs.

AMPAR block triggered non-uniform AMPAergic scaling. Scaling ratio plot shows the ratio of ran ordered mEPSC amplitudes from CNQX-treated culture (n=95 cells, 91mEPSCs/cell) divided by those from untreated cultures (n=91 cells, 95 mEPSCs/cell). The X axi represents the rank ordered number of mEPSCs (from smallest to largest).

Custom written Matlab program identifies bursts in cortical cultures plated on MEA’s by choosing the minimum number of spikes per burs (Spikes/Burst) across a minimum number of channels contributing to a burst (Min channels) within a maximum Time Window. Upper image shows the identification of burst in red across 64 channels as a raster plot where each dot represents one spike detected on the MEA. The program then examines various parameters which were then exported to an excel spreadsheet for analysis. Burst identitiy and duration are shown as a red line positioned below the raster plot A single burst is expanded and plotted below the upper image.

Rasterplot of cortical culture plated on MEA demonstrating network bursting (red dots, upper plot). Bursts were then abolished after addition of TTX (1μM) to theculture; a small number of spike detections remain, however these are likey to be noise that crosses the detection threshold.

MEA recordigns show optostim + CNQX increases burst frequency and spike frequency compared to CNQX alone. A-B) The average Burst rate (A) or spike frequency (B) is shown in cultures following CNQX + photostim or just CNQX over 24 hrs, The mean differences at different time points are compared to control and displayed in Cumming estimation plots. Significant differences denoted by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Recordings from single cultures (filled circles), where mean values (represented by the gap in the vertical bar) and SD (vertical bars) are plotted on the upper panels. Mean differences between control and treated groups are plotted on the bottom panel, as a bootstrap sampling distribution (mean difference is represented by a filled circles and the 95% Cls are depicted by vertical error bats).