Article Figures & Data
Figures
- Figure 1.
Firing rate characteristics analyzed in cortical cultures. Raster plot of network burst activity across all channels in the MEA. Interburst interval (IBI) spike rate is the spiking that occurred outside of the bursts (spikes are black dots). SRWB is the spike rate that occurs within the burst (spikes are red dots). Burst duration is shown by the red lines below raster plot. Extended Data Figure 1-1 provides more details on the program that analyzes bursts.
- Figure 2.
Burst dynamic parameters measured following GABAR blockade (bicuculline) in cortical cultures. A, Normalized overall spike rate, (B) normalized burst frequency, (C) normalized interburst interval (IBI) spike rate and (D) normalized burst duration displayed over a 24 h period for control and bicuculline-treated cultures. Values at each time point are normalized to baseline (predrug) condition. Each color line represents a single culture, with the thick black line representing the mean of all cultures. The data shown in A and B are the same data as in our previous publication (Gonzalez-Islas et al., 2024); however, here we follow individual cultures over time. Estimation statistics comparing bicuculline-treated and control cultures at each of the time points establish that none are significantly different. This is likely due to the dramatic variability. Extended Data Figure 2-1 provides estimation statistics for data shown in Figure 2.
- Figure 3.
Spike rate within a burst (SRWB) following GABAR blockade is consistently homeostatically recovered in cortical cultures. A, B, SRWB displayed over a 24 h period for (A) control (untreated) and (B) bicuculline-treated cultures. Values at each time point are normalized to baseline (predrug) condition. Each color line represents a single culture, with the thick black line representing the mean of all cultures. C, SRWB is compared for control and bicuculline-treated cultures at 0.5, 1, 3, 6, and 24 h after addition of bicuculline. The mean differences at different time points are compared with control and displayed in Cumming estimation plots. Significant differences denoted by *p < 0.05, ***p < 0.001. The top panel shows raw data from single culture recordings (filled circles), where the mean value is represented by the gap in the vertical bars and the SD is represented by the vertical bars. The bottom panel shows mean differences between control and treated groups as a bootstrap sampling distribution (mean difference is represented by filled circle and the 95% CIs are depicted by vertical error bars). Extended Data Figure 3-1 graphs non-normalized values for each culture individually. Extended Data Figure 3-2 shows non-normalized values for three cultures treated with gabazine instead of bicuculline.
- Figure 4.
Homeostatic recovery percentage for different firing rate properties. The homeostatic index for each bicuculline-treated culture was calculated by dividing the actual recovery by the full potential recovery (see text). Horizontal line represents 100% recovery. Spike frequency within a burst is the firing rate property that most consistently demonstrates near 100% homeostatic recovery.
- Figure 5.
Spike rate within a burst (SRWB) following GABAR blockade was homeostatically recovered at both the population and single-unit level. A, In control cultures, the number of MEA channels contributing to a burst remained stable. B, Following bicuculline, the number of MEA channels contributing to a burst increased and was then homeostatically restored over a 24 h period. Values at each time point were normalized to the baseline (predrug) condition. Each color line represents a single culture, with the thick black line representing the mean of all cultures. C, Following bicuculline, SRWB for most individual sorted units increased and were homeostatically restored over a 24 h period. Values at each time point were normalized to the baseline (predrug) condition. Each line represents a single unit from one of four cultures (units from same cultures are of the same color), with the thick black line representing the mean of all units. Inset: one (C1) or two (C2) waveforms were identified on two different channels (SD shown in shaded area).
- Figure 6.
Firing rate characteristics analyzed in the isolated embryonic chick spinal cord. A, Schematic of 32-channel NeuroNexus probe inserted into the spinal cord, specifically penetrating the motor column. B, Raster plot of network burst and episode activity across channels on the NeuroNexus probe. Episodes caught by the custom-written Matlab program are highlighted in blue, with spikes caught in the episodes as red dots. Purple lines below the episodes represent where bursts occurred within the episode. Black dots are spikes that occurred outside of the episode. Thus, the interepisode interval spike rate is calculated by taking the spike rate outside of the episodes. C, The raster is zoomed into the last episode from A. The red dots again represent the spikes within the episode and the purple lines denote the bursts. From this data, we can calculate the episode spike rate, which includes all spikes within the episode, regardless of whether or not the spikes were part of a burst. D, The raster is zoomed into the last burst from B. We calculated the burst duration and spike rate within the burst within episodes from this data. Extended Data Figure 6-1 provides MEA recordings showing waveforms showing motoneuron antidromic activation.
- Figure 7.
Spiking parameters measured following GABAR blockade (gabazine) in embryonic chick spinal cords. A, Normalized overall spike rate, (B) normalized interepisode interval (IEI) spike rate, and (C) normalized burst duration displayed over a 3 h period for control (untreated) and gabazine-treated cords. Each color represents a single cord, with the height of the bar representing the mean of all cords. Estimation statistics comparing gabazine-treated and control cords at each of the time points establish that none were significantly different in B or C. In A, significant p values were observed for all comparisons—p < 0.001 for 0–30, 30–60, 60–90, and 150–180 min comparisons and p < 0.01 for 90–120 and 120–150 min comparisons. Extended Data Figure 7-1 provides estimation statistics for data shown in Figure 7. Extended Data Figure 7-2 shows episode frequency. Extended Data Figure 7-3 shows episode spike rate and duration.
- Figure 8.
Spike rate within a burst (SRWB) within episodes following GABAR blockade in the isolated spinal cord was consistently homeostatically recovered. A, B, SRWB displayed over a 3 h period for (A) control (untreated) and (B) gabazine-treated cords. Values in each 30 min bin are normalized to baseline (predrug) condition. Each color represents a single spinal cord, with the height of the bar representing the mean of all cords. C, SRWB is compared for control and gabazine-treated cords at 30 min intervals during the 3 h recording period. The mean differences at each time point are compared with control and displayed in Cumming estimation plots. Significant difference is denoted by *p < 0.05. The top panel shows raw data from single spinal cord recordings (filled circles), where the mean value is represented by the gap in the vertical bars and the SD is represented by the vertical bars. The bottom panel shows mean differences between control and treated groups as a bootstrap sampling distribution (mean difference is represented by filled circle and the 95% CIs are depicted by vertical error bars).
- Figure 9.
Calcium imaging of gabazine-treated embryonic chick spinal cords. A, Motor neurons were labeled with Calcium Green Dextran overnight. Scale bar, 100 μm. B, Results show that after gabazine addition there was an initial increase in calcium fluorescence transients associated with episodes followed by a recovery to baseline levels. Episodes were stimulated and calcium transients were recorded for three spinal cords (black line, average).
Tables
- Table 1.
Mean and standard deviation of all spiking activity features that were analyzed for culture preparations
Feature Prebicuculline value Overall spike rate 42.51 ± 55.36 Burst frequency 0.24 ± 0.24 Interburst interval spike frequency 8.14 ± 8.21 Burst duration 1.07 ± 2.23 Spike rate within a burst 166.27 ± 76.46 Number of channels in a burst 16.24 ± 14.11 All features are reported in Hz, except for burst duration (seconds) and the number of channels.
- Table 2.
Coefficient of variation (CV) and mean for homeostatic indices of all spiking activity features that were analyzed for culture preparations and shown in Figure 4
Feature CV Mean SRWB 0.19 90.32 Burst duration 0.86 46.65 Overall spike rate −7.12 −36.56 Interburst interval spike rate −46.22 −3.32 Burst frequency 0.99 73.75 All features are reported in Hz, except for burst duration and episode duration (seconds).
- Table 3.
Mean and standard deviation of all spiking activity features that were analyzed for spinal cord preparations
Feature Pregabazine value Overall spike rate 0.19 ± 0.06 Interepisode interval spike frequency 0.01 ± 0.01 Burst duration 0.09 ± 0.02 Episode spike rate 0.13 ± 0.04 Episode duration 42.74 ± 8.16 Spike rate within a burst 1.72 ± 0.71 All features are reported in Hz, except for burst duration and episode duration (seconds).
Figure 1-1
Program that detects and analyzes bursts. A) Probability distribution of ISI durations between 10 consecutive spikes (n to n + 9) largest dip in the distribution (200 ms) is used to detect bursts in the burst detection subroutine of the program. B) Bust detection program identifying bursts that contain at least 10 spikes in 200 ms. Burst duration is shown in red line below raster plot. Burst spikes are red and spikes in the inter-burst interval are black. Download Fig 1-1, TIF file.
Figure 2-1
Estimation statistics of burst dynamic parameters from bicuculline-treated cultures for A) overall spike rate B) burst frequency C) inter-burst interval (IBI) spike rate and D) burst duration. The mean differences at each time point are compared to control and displayed in Cumming estimation plots. Upper panel shows raw data from recordings of individual cultures (filled circles), where the mean value is represented by the gap in the vertical bars and the SD is represented by the vertical bars. Lower panel shows mean differences between control and treated groups as a bootstrap sampling distribution (mean difference is represented by filled circle and the 95% CIs are depicted by vertical error bars). Download Fig 2-1, TIF file.
Figure 3-1
Nine different cortical cultures show that addition of bicuculline triggers an increase in SRWB, which then is homeostatically returned to baseline levels. SRWB is calculated as the average SRWB across individual bursts and standard deviation of SRWB is shown as error bars. Data is not normalized and therefore represents the MEA-wide spike rate within a burst of each culture before (black) and after bicuculline (red). Download Fig 3-1, TIF file.
Figure 3-2
Three different cortical cultures show that addition of gabazine triggers an increase in SRWB, which then is homeostatically returned to baseline levels. SRWB is calculated as the average SRWB across individual bursts and standard deviation of SRWB is shown as error bars. Data was not normalized and therefore represents the MEA-wide spike rate within a burst for each culture before (black) and after gabazine (red). Download Fig 3-2, TIF file.
Figure 6-1
Antidromic stimulation to identify embryonic chick spinal cord motor neurons. A) Asterisks mark the component that identifies motor neurons in the raw trace. B) No motor neuron component is present in the deeper channels/interneurons. Download Fig 6-1, TIF file.
Figure 7-1
Estimation statistics of firing dynamic parameters following GABAR blockade (gabazine) in spinal cords for A) overall spike rate B) inter-episode interval (IEI) spike rate and C) burst duration. The mean differences at each time point were compared to control and displayed in Cumming estimation plots. Significant differences denoted by ** p < 0.01, *** p < 0.001. Upper panel shows raw data from single spinal cord recordings (filled circles), where the mean value is represented by the gap in the vertical bars and the SD is represented by the vertical bars. Lower panel shows mean differences between control and treated groups as a bootstrap sampling distribution (mean difference is represented by filled circle and the 95% CIs are depicted by vertical error bars). Download Fig 7-1, TIF file.
Figure 7-2
Episode frequency following GABAR blockade in the isolated spinal cord is not homeostatically recovered. Frequency of episodes in spinal cords is displayed as the time interval from the last episode. Each color dot represents a single spinal cord. A) Episode frequency of control cords. B) Episode frequency of gabazine-treated cords. Gabazine was added at time point 0 seconds. Download Fig 7-2, TIF file.
Figure 7-3
Episode spike rate and episode duration following GABAR blockade were variable in both control and gabazine-treated spinal cords. A) Episode spike rate displayed over a 3-hour period for control (untreated) and gabazine-treated cords. Values in each 30-minute bin are normalized to baseline condition. Each color dot represents a single spinal cord, with the height of the bar representing the mean of all cords. B) Episode duration displayed over a 3-hour period for control (untreated) and gabazine-treated cords. Values in each 30-minute bin are normalized to baseline condition. Each color dot represents a single spinal cord, with the height of the bar representing the mean of all cords. Download Fig 7-3, TIF file.
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