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 (red rectangle) is expanded in time and shown in the raster plot on the right. B) The normalized burst rate is shown in control cultures and following application of CNQX for 24 hrs. C) Average overall spike frequency is compared for CNQX-treated cultures and control unstimulated cultures at 1hr, 3hrs, 6hrs (p=0.104), and 24hrs (p=0.982) after addition of CNQX or vehicle. 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, control n = 3 cultures, CNQX n = 8 cultures), 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 mIPSC amplitudes that appear to scale down. A) CNQX and TTX produce a reduction in average amplitude of mIPSCs as shown in the scatter plots (control - n=21 from 10 cultures, TTX - n=7 from 3 cultures, CNQX - n=10 from 6 cultures). 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% CIs are depicted by vertical error bars). Example traces showing mIPSCs are shown below. B) Scaling ratio plots show the relationship of mIPSC 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-occurring 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) Average burst rate is compared for CNQX-treated cultures with optogenetic stimulation (n=5 cultures) and control unstimulated cultures (n=3 cultures) at 1hr, 3hrs, 6hrs (p=0.056), and 24hrs (p=0.379) after addition of CNQX or vehicle (same control data presented in Figure 1). D) Average overall spike frequency is compared for CNQX-treated cultures with optogenetic stimulation and control unstimulated cultures at 1hr (p=0.612), 3hrs (p=0.489), 6hrs (p=0.449), and 24hrs (p=0.22) after addition of CNQX or vehicle. Control data is same as presented in Figure 1. 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 difference is represented by a filled circles and the 95% CIs 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 plots show AMPAR blockade triggers a reduction in mIPSC amplitude compared to controls that is prevented when combined with optogenetic stimulation (optostim, control - n=16 from 10 cultures, CNQX - n=8 from 4 cultures, CNQX/optostim - n=13 from 6 cultures). The mean differences are compared to control and displayed in Cumming estimation plots. Significant differences denoted by ** p ≤ 0.01, *** p ≤ 0.001. GABAergic mIPSC 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% CIs 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 CTZ and this was dependent on spiking activity. A) MEA recordings show that CTZ-treated cultures trended toward increases in normalized burst rate compared to control untreated cultures at 1hr (p=0.97), 3hrs (p=0.246), 6hrs (p=0.397), and 24hrs (p=0.894) after addition of CNQX (n=7) or vehicle (n=5). B) MEA recordings show that CTZ-treated cultures trended toward increases in normalized overall spike rate compared to control untreated cultures at 1hr (p=0.565), 3hrs, 6hrs (p=0.634), and 24hrs (p=0.92) after addition of CNQX or vehicle. C) Control cultures in Neurobasal (nrbsl) were compared with control cultures with ethanol (EtOH) dissolved in Neurobasal (1:1000). Amplitude of mIPSCs in different controls were no different (p=0.803, nrbsl - n=21 from 10 cultures, EtOH - n=11 from 3 cultures). D) CTZ treatment (dissolved in ethanol) led to an increase in mIPSC amplitude compared to ethanol control cultures (CTZ - n=8 from 3 cultures). CTZ combined with TTX (in ethanol) produced a reduction of mIPSC amplitude compared to controls (ethanol) that was no different than TTX (nrbsl) alone (CTZ+TTX - n=7 from 3 cultures, TTX - n=7 from 3 cultures is same data as shown in Figure 2A). 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. 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. A) Bicuculline-treated cultures (24hrs) plated on MEA’s trended upward in normalized burst rate compared to control untreated cultures at 1hr (p=0.314), 3hrs (p=0.246), 6hrs (p=0.339), and 24hrs (p=0.339) after addition of bicuculline (n=9 cultures) or vehicle (n=3 cultures, same data as Figure 1). B) Bicuculline-treated cultures (24hrs) plated on MEA’s trended upward in normalized overall spike frequency compared to control untreated cultures at 1hr (p=0.358), 3hrs (p=0.462), 6hrs (p=0.734), and 24hrs (p=0.772) after addition of bicuculline or vehicle. 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 mIPSC amplitudes (control n=21 form 10 cultures, bicuculline - n=10 from 4 cultures). The mean difference is compared to control and displayed in Cumming estimation plots. Significant difference denoted by * p ≤ 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% CI is depicted by vertical error bar). D) Ratio plots for bicuculline-induced increase in mIPSCs exhibits a multiplicative profile. All mIPSC amplitudes recorded from cultures plated on coverslips, not MEAs.

AMPAergic scaling was absent following 24 hours of 20µM CNQX. AMPA mEPSC amplitudes were no different than control following AMPAR blockade (p=0.57, control - n=9 from 4 cultures, CNQX - n=8 from 3 cultures). Recordings from single neurons (filled circles), where mean values (represented by horizontal bar) are plotted, as a bootstrap sampling distribution (mean difference is represented by a filled circles and the 95% CIs are depicted by vertical error bars). All mIPSC amplitudes recorded from cultures plated on MEAs.

AMPAR block triggered non-uniform AMPAergic scaling. Scaling ratio plot shows the ratio of rank ordered mEPSC amplitudes from CNQX-treated cultures (n=95 cells, 91mEPSCs/cell) divided by those from untreated cultures (n=91 cells, 95 mEPSCs/cell). The X axis 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 burst (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 bursts 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 identity and duration are shown as a red line positioned below the raster plot. A single burst is expanded and plotted below the upper image.

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

MEA recording’s show optostim + CNQX increases burst frequency and spike frequency compared to CNQX alone. A) Average burst rate is compared for CNQX-treated cultures with optogenetic stimulation (n=5) and CNQX only unstimulated cultures (n=8) at 1hr, 3hrs, 6hrs, and 24hrs (p=0.209) after addition of CNQX. B) Average overall spike frequency is compared for CNQX-treated cultures with optogenetic stimulation and CNQX only unstimulated cultures at 1hr, 3hrs, 6hrs, and 24hrs (p=0.389) after addition of CNQX. 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).

Frequency of mIPSCs were no different across conditions. Scatterplots of mIPSC frequency show tremendous variability but do not exhibit significant differences through different drug treatments. The mean differences are compared to control and displayed in Cumming estimation plots. GABAergic mPSC frequencies 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% CIs are depicted by vertical error bars).