Multi-electrode recording of spontaneous activity in a dissociated cortical culture. (A) Typical three week old culture on a multi-electrode array (MEA). (B) Bursts were classified into two categories: small and big bursts depending on the threshold number of spikes within a burst. The figure shows a histogram of the number of spikes in each burst from three of the cultures used. The red line indicates the threshold for each culture. Note that the axis scales are different for each culture. (C) Spike raster plot and array-wide spike rate histograms of spontaneous activity for 5 min of spontaneous recording. The inset shows typical examples of bursts classified as ‘big’ and ‘small’ bursts. Recording was taken at 24 days in vitro.

Shuffled data do not show a significant structure in the BAM of spontaneous bursts. (A) Sorted correlation matrix of actual unshuffled data for a representative tetanus experiment. There were high correlations indicating a high similarity among the BAM of spontaneous bursts. (B) Average correlation values between the BAM clusters from N = 5 tetanus experiments after different shuffling methods: ‘electrode shuffle’, ‘temporal shuffle’, ‘matched shuffle’, ‘spike swapping’ and ‘spike jittering’ with 10 and 20 ms standard deviations. The average correlation of the original data showed significantly higher correlation values compared to shuffled data (the asterisk indicates p < 0.001, Wilcoxon's rank sum test for equal medians).

Examples of clusters for the BAM of spontaneous bursts for the experiment of figures 4(A) and (B). The sampled times are marked by arrows in figure 4. A given cluster shows similarity in the spatiotemporal structure over hours, but there was an easily visible difference between the patterns of any two clusters. (A) Frames from two representative BAM clusters for small bursts are shown, in the original coordinates of the multi-electrode array, at 20 ms intervals. (B) Frames from two representative BAM clusters for big bursts are shown, in the original coordinates of the multi-electrode array. Frames are shown at 100 ms intervals since the big bursts were much longer than the small bursts shown in (A). The sizes of the circles represent the number of spikes in that particular electrode for a particular time bin.

Pictorial description of the analysis of spatiotemporal patterns within spontaneous bursts and statistics on the change of distribution of burst patterns across the tetanus. (A) Raster plot and corresponding histogram of spontaneous spiking activity on 60 electrodes. Small and big bursts were detected, the onset and offset times of bursting activity were calculated and the difference between the offset and onset times was determined as the length of the burst. The burst activity matrix (BAM) for each burst was generated by counting the number of spikes within the length of the burst on each of the 60 electrodes using a 100 ms moving time bin (time step = 10 ms). The right panel shows frames of a BAM for different time bins for 60 electrodes (arranged in the original coordinates of the multi-electrode array). The size of the circle represents the number of spikes on that particular electrode for a particular time bin with the colored circle representing the value at electrode k, and the firing rate time histogram for electrode k is shown below. The BAM for each burst was then constructed by appending the histograms from the 60 recording electrodes, together to form a 1 by 60 N matrix, where N is the number of time bins in the longest burst. (B) A two-dimensional cross-section of a BAM plotted in 60 by N dimensional space is shown. BAMs of all bursts were compared against each other and clustered using a paired clustering algorithm (dendrogram, see supplementary materials stacks.iop.org/PhysBio/4/181). Occurrence represents when a burst occurs within a specific BAM cluster (figures 3 and 4). Integrated occurrence was defined as the total number of occurrences of bursts in a 15 min time window moved by a time step of 30 s. Integrated occurrences were calculated across three periods of the same length equally spaced in time, Pre1, Pre2, Post1, and Post2. Periods Pre1 and Pre2 were before the tetanus stimulation and periods Post1 and Post2 were after the tetanus. (C) A two-dimensional cross-section of integrated occurrences of all BAM clusters (S by R dimensional matrix, S = number of BAM clusters, R = number of 15 min moving time windows) is shown. The color bar represents the time elapsed during the experiment and each point represents the integrated occurrence of the BAM clusters. The centroids of integrated occurrences in periods Pre1, Pre2 and Post1 were calculated. The change across periods was calculated as the Euclidean distance of each point in a period from the centroid of the previous period (d₁₂, d₂₃ and d₃₄ in the figure) normalized by the Euclidean distance of each point in the previous period from its centroid (d₁₁, d₂₂ and d₃₃ in the figure). This calculation yielded three quantities: C/D before the tetanus (Pre1–Pre2), C/D across the tetanus (Pre2–Post1) and C/D after the tetanus (Post1–Post2). The difference between two C/Ds from consecutive periods was tested (Wilcoxon's rank sum test) to evaluate changes in the burst pattern across periods.

Occurrence of spontaneous bursts was stable before the tetanus and changed across the tetanus. (A) Each stroke represents one occurrence of a BAM cluster of small spontaneous bursts. (B) Each stroke represents one occurrence of a BAM cluster of big spontaneous bursts. This is a representative example from one experiment. The gray bar indicates the period of tetanization. The arrowheads indicate the timing of the different BAM clusters shown in figure 5. (C) In five experiments, the average change in integrated occurrences across the tetanus (C/D between Pre2 and Pos t1) was significantly greater than the average drift before the tetanus (C/D between Pre1 and Pre2) (p < 0.01). C/D = 1 indicates that there was no change in the occurrence between periods. C/D before the tetanus (Pre1–Pre2) was close to 1, indicating that there was very little drift in the Pre period before the tetanus and the integrated occurrence of the BAM of spontaneous bursts was stable for this period. **indicates p < 0.01 and * indicates p < 0.05 (Wilcoxon's rank sum test for equal medians).

Burst initiation sites were significantly changed by a strong tetanus. (A) The probability (burst initiation probability, BIP) of a recorded neuron having initiated a burst is shown by the different sizes of the circles for a representative experiment. Although this probability did not change much in the spontaneous recording periods before the tetanus (Pre1 and Pre2), it changed significantly across the tetanus. (B) The average change in the probability of burst initiation for all experiments changed significantly after (p < 0.05) but not before the tetanus (N = 5 experiments). The asterisk indicates p < 0.05, Wilcoxon's rank sum test. The error bars represent SEM.