GABAergic synaptic scaling is triggered by changes in spiking activity rather than AMPA receptor activation
- Department of Cell Biology, Emory University, United States
- Doctorado en Ciencias Biológicas Universidad Autónoma de Tlaxcala, Mexico
- Department of Neurosurgery, Emory University, United States
- Department of Biomedical Engineering, Georgia Tech and Emory University, United States
- Department of Neuroscience, Cell Biology and Physiology, Wright State University, United States
Figures
Figure 1 with 2 supplements
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 multi-electrode array (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 hr. (C) Average overall spike frequency is compared for CNQX-treated cultures and control unstimulated cultures at 1 hr, 3 hr, 6 hr (p=0.104), and 24 hr (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).
Figure 1—figure supplement 1
Custom-written MATLAB program identifies bursts in cortical cultures plated on multi-electrode arrays (MEAs) 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.
Figure 1—figure supplement 2
Rasterplot of cortical culture plated on multi-electrode array (MEA) demonstrating network bursting (red dots, upper plot).
Bursts were then abolished after addition of TTX (1 µM) to the culture; a small number of spike detections remain, however these are likely to be noise that crosses the detection threshold.
Figure 2 with 1 supplement
Both activity and AMPA receptor (AMPAR) blockade cause a reduction in miniature inhibitory postsynaptic current (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 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 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 multi-electrode arrays (MEAs).
Figure 2—figure supplement 1
AMPA receptor (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, 91 mEPSCs/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).
Figure 3 with 1 supplement
Multi-electrode array (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 two 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 1 hr, 3 hr, 6 hr (p=0.056), and 24 hr (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 1 hr (p=0.612), 3 hr (p=0.489), 6 hr (p=0.449), and 24 hr (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).
Figure 3—figure supplement 1
Multi-electrode array (MEA) recordings 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 1 hr, 3 hr, 6 hr, and 24 hr (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 1 hr, 3 hr, 6 hr, and 24 hr (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).
Figure 4
Optogenetic restoration of spiking activity in the presence of AMPA receptor (AMPAR) blockade prevents GABAergic downscaling observed in CNQX alone.
(A) Scatter plots show AMPAR blockade triggers a reduction in miniature inhibitory postsynaptic current (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 + optostimulation treatments. Cultured neurons for these recordings were obtained from cells plated on multi-electrode arrays (MEAs) (control, CNQX, and CNQX + optostim).
Figure 5
GABAergic upscaling was triggered by cyclothiazide (CTZ) and this was dependent on spiking activity.
(A) Multi-electrode array (MEA) recordings show that CTZ-treated cultures trended toward increases in normalized burst rate compared to control untreated cultures at 1 hr (p=0.97), 3 hr (p=0.246), 6 hr (p=0.397), and 24 hr (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 1 hr (p=0.565), 3 hr, 6 hr (p=0.634), and 24 hr (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 miniature inhibitory postsynaptic currents (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.
Figure 6 with 1 supplement
GABAergic upscaling is triggered by increased spiking activity rather than reduced GABAR activation.
(A) Bicuculline-treated cultures (24 hr) plated on multi-electrode arrays (MEAs) trended upward in normalized burst rate compared to control untreated cultures at 1 hr (p=0.63), 3 hr (p=0.556), 6 hr (p=0.547), and 24 hr (p=0.559) after addition of bicuculline (n=9 cultures) or vehicle (n=3 cultures, same data as Figure 1). (B) Bicuculline-treated cultures (24 hr) plated on MEAs trended upward in normalized overall spike frequency compared to control untreated cultures at 1 hr (p=0.358), 3 hr (p=0.462), 6 hr (p=0.734), and 24 hr (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 (24 hr) produced an increase in miniature inhibitory postsynaptic current (mIPSC) amplitudes (control - n=21 from 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 exhibit a multiplicative profile. All mIPSC amplitudes recorded from cultures plated on coverslips, not MEAs.
Figure 6—figure supplement 1
Frequency of miniature inhibitory postsynaptic currents (mIPSCs) was no different across conditions.
Scatter plots of mIPSC frequency show tremendous variability but do not exhibit significant differences through different drug treatments. The mean differences are compared to their respective controls and displayed in Cumming estimation plots. (A) The miniature postsynaptic current (mPSC) frequencies from cultures plated on coverslips were no different from controls in any of the drug conditions, including CNQX (p=0.243), TTX (p=0.301), bicuculline (p=0.186), and ethanol (p=0.201). (B) The mPSC frequencies from cultures plated on multi-electrode arrays (MEAs) were no different from controls after CNQX (p=0.826) or CNQX + photostimulation (p=0.773). (C) The mPSC frequencies from cultures plated on coverslips were no different from controls after cyclothiazide (CTZ) (p=0.827) or CTZ + TTX (p=0.301). 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).
Figure 7
AMPAergic scaling was absent following 24 hr of 20 µM CNQX.
AMPA mEPSC amplitudes were no different than control following AMPA receptor (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 miniature excitatory postsynaptic current (mEPSC) amplitudes recorded from cultures plated on multi-electrode arrays (MEAs).
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GABAergic synaptic scaling is triggered by changes in spiking activity rather than AMPA receptor activation
eLife 12:RP87753.
https://doi.org/10.7554/eLife.87753.3
