3.1. Optimal spike detection threshold
We chose to detect spikes as events whose voltage extremes exceeded a threshold within a 2 ms window, set as a multiple of the standard deviation (SD) of the noise. To optimize, we detected spikes using thresholds varying from 8 to 13 SD, followed by a repeated measure on the same network after inhibition of synaptically induced spiking activity and low level biological noise with an APV/CNQX/TTX cocktail to block NMDA and AMPA receptors and sodium channels, respectively. We measured the difference in the number of apparently active electrodes before and after drug application at each threshold setting, looking for the maximum difference (), finding that a multiplier of 11 was optimal. Further we measured the ratio of total spikes detected after to before drug application, looking for a near zero asymptote (the lowest SD that was not significantly different from zero), which occurred at 11 SDs. Thus, a setting of 11 SD permits a maximal number of evoked channels and spikes to be detected, but a minimal number of operationally false positive noisy or indirectly active channels and events. We note this is approximately equivalent to a zero to peak threshold of 5.5 x S.D. for a symmetric bipolar spike.
Pharmacological analysis suggests that 11 x S.D. peak to peak is the best threshold for spike detection
3.2. Effects of chronic stimulation and distance from stimulus on active channels
provides an example of the average number of channels that evoked a response to the stimulus (S). shows that an average of 2.7 channels per stimulus was activated for each distance (200–1721 μm), which suggests that 16 channels on average were activated every time a stimulus was delivered, averaged over all chronic stimulation conditions. Based on all arrays tested, shows that the fraction of channels recording any evoked activity during the entire probe recording (~17 min) was 45–50% with no significant increase due to chronic stimulation. In contrast, this response rate was two-fold higher than spontaneous activity without chronic stimulation and 30% higher than spontaneous activity with chronic stimulation (Brewer et al., 2009b
). The percent active electrodes could surely be increased by increasing the density of plated neurons or by addition of extra astroglia (Boehler et al., 2007
Average number of active channels does not change with either chronic stimulation or distance from the stimulus
3.3. Effects of chronic stimulation and distance from stimulus on evoked activity
shows the neuron distribution on a MEA at 21 DIV cultured in NbActiv4 medium, for each condition (0, 1 and 3 hr/day chronic stimulation). Chronic stimulation did not change their morphological properties or cause neuron death. also provides examples of evoked activity recorded 2–40 ms after the stimulus was delivered to electrodes located close (283 μm) and far (1721 μm) away from the stimulation site. Notice three parameters changed with distance from the stimulus: i) the probability of an evoked spike decreased, ii) latencies increased and iii) evoked spike amplitudes decreased.
In comparison to the unstimulated condition, chronic stimulation increases the probability of response without changing morphological properties of neurons cultured on MEAs
We first determined how the Euclidian distance from a recording to a stimulating electrode affects the probability of a response to the stimulus. Note that the distances between closest, diagonally adjacent, and most widely separated pairs of electrodes are 200 μm, 283 μm and 1721 μm, respectively (). Chronic stimulation (1 or 3 hours) led to higher response rates (50% or 35% vs. no chronic stimulation) at recording electrodes near the probe electrode (≤283 μm away) (). Response rates decreased 3 fold and monotonically with distance to the furthest electrode (). Electrodes in close proximity to the stimulus (283 μm) recorded 3-fold more spike responses than the ones at longest distance (1721 μm). During 1 hr/day chronic stimulation, the number of spikes per stimulus, for all distances, was 10–15% higher than with no chronic stimulation, with the values for 3 hr/day stimulation falling in between. The results show both enhancements due to chronic stimulation and connectivity that declines with distance.
Probability of an evoked response increases with chronic stimulation and decreases with distance from the stimulus
3.4. Effects of chronic stimulation and distance from stimulus on first spike latency
We tested the hypothesis that neurons in close proximity to the stimulus tend to be activated more often than the distant ones, consequently evoking more spike responses. illustrates that, near the stimulating electrode, it is common to record spikes at short latency and uniform amplitude, suggesting reliable, direct or at most monosynaptic excitation of the same neuron. In contrast, at maximal distance, shows a variety of spike latencies and amplitudes, suggesting activation of different paths and recorded neurons. shows that the coefficient of variation of the latency (mean/SD) increases with distance (from 0.8 to 1.6), largely independent of time of chronic stimulation, indicating greater dispersion with distance from the stimulus site. The latencies summarized in suggest near linear propagation with distance, equivalent to a speed of 0.1 mm/msec. If one extrapolates to zero distance, the latency is 1.2 msec, which may be the time to activation of a directly stimulated neuron. Alternatively, the graph appears to asymptote at 4 msec, the same as might be inferred as the time to activation of a directly responding neuron (Bakkum et al. 2008
). Therefore, at maximum distance of 1721 μm, an average delay of 16 ms suggests either 3 or 4 synapses.
First spike latency increases with distance from the stimulus
3.5. Effects of chronic stimulation and distance from stimulus on spike amplitude evoked by the 2nd pulse
Even if extracellular recordings are not the best choice to assess changes in amplitude, we examined evidence for spike amplitude plasticity because the developmental accumulation of sodium channels at the axon initial segment might be influenced by chronic stimulation. We also know that our recordings come from multiple neurons, so any change in amplitude could be from neurons with different identities. Spike sorting was considered problematic due to the huge variety of spike shapes observed in our cultures, especially for overlapping waveforms during bursts, as pointed out also by others (Eytan and Marom, 2006
; Rolston et al., 2007
; Chiappalone et al., 2008
To test for spike amplitude plasticity caused by paired pulse or chronic stimulation, we compared first spike latency and amplitude responses from the 1st to those from the 2nd pulse. We found no differences in the probability of the number of responses or spike latency (data not shown). However, summarizes a remarkable 4-fold increase in the evoked spike amplitude of the 2nd pulse minus that evoked by the 1st pulse.
The 2nd pulse increases spike amplitudes during chronic stimulation
To determine whether distance factored into this change in spike amplitude, shows examples of responses from a chronically stimulated culture for each of the paired pulses at distances close and far from the stimulating electrode. In this case, notice that near the stimulus, spike amplitudes to the paired pulses are similar (Bi-ii), and that far away the spike amplitude is much larger for the 2nd pulse (Biv) compared to the 1st pulse (Biii). Our observations are summarized in , indicating the general decline in amplitude with distance. Remarkably, the condition of 3 hr/day chronic stimulation (7E) resulted in an enhancement of the amplitude of the initial spikes to the 2nd vs. the 1st of the probing pulse pairs. Indeed, the 2nd pulse response did not decline with distance. shows the histogram for the evoked spike amplitudes. For amplitudes lower than 50 μV, both distributions were similar; however, the 2nd pulse elicited more responses with higher amplitudes (see the insets). Several possible mechanisms could explain the enhancement in spike amplitude by the 2nd pulse: a) spillover of a burst elicited by the 1st pulse into the recorded response to the 2nd stimulus; b) different neurons being recruited each time; c) compound action potentials; or d) spike amplitude plasticity.
3.6. Spikes are larger in bursts than isolated spikes: spike spillover
To determine which of these mechanisms was responsible for the spike amplitude increases, we examined individual responses to stimuli. Figure 8Ai-ii shows an example of a delayed burst evoked by the 1st pulse at 1721 μm distance. At this distance, average latency was 16 ms (). Therefore, we chose a window of latencies longer than 10 ms for the 1st pulse and shorter than 10 ms for the 2nd pulse to examine the responses to the 2nd pulse that could be spillover of burst firing into the 2nd window. We found that in response to the 1st pulse, 65% of the records contained spikes after 10 ms, which could possibly spillover into the window of the 2nd pulse. Also, in response to the 2nd pulse, 45% of the records evoked a spike before 10 ms. These large percentages suggest a high frequency of burst continuation or spillover from the 1st to the 2nd window. If the first spike after a pulse occurs during a burst, its amplitude is likely to vary considerably according to the identity of the neuron, position of the spike within the burst, and partial or complete overlap of spikes from the 1st pulse, complicating interpretation. These factors precluded further analysis but indicate a large fraction of spikes during the time window following the 2nd pulse were due to spillover of burst activity evoked by the 1st pulse.
If burst spikes evoked by the 1st
stimulus spillover into the time window of the 2nd
stimulus, then spike frequency might increase in the window of responses to the 2nd
pulse. Since spontaneous burst duration was 300 ms in these same networks (Brewer et al., 2009b
), if a burst was initiated during the 50 ms following the 1st
pulse, it would likely continue or spillover into the window that follows the 2nd
pulse. One characteristic of a burst is a higher spike rate. In , we calculated the inter-spike interval (ISI) of evoked responses and represented it in terms of an instantaneous firing rate (1/ISI). Note that the frequency is highest for the 3 hr/day condition, indicating a higher spike rate within bursts for this chronic stimulation. , shows a stimulation-dependent distribution of firing rates with rates higher than 600 Hz only for the 3 hr/day condition.
Firing rate and spike amplitudes following the 2nd pulse are higher due to spillover from evoked bursts with higher amplitudes than those of isolated spikes
We next determined whether chronic stimulation affected spike amplitude either for isolated spikes or spikes within a burst. The spontaneous activity from the same arrays (Brewer et al., 2009b
), exhibited a mean intra-burst frequency of 50 Hz, about 2 spikes in our 2–40 ms window for the evoked activity. Considering this criterion for possibly evoked ‘bursts’, in we sorted evoked responses into singles (1 spike only, 60%) and ‘bursts’ (≥2 spikes, 40%). We found lower spike amplitudes in the single response category, medium spike amplitudes in mid-burst (all spikes but the first) and largest spike amplitudes in the first spike of a burst. This decrease in mid-burst spike amplitudes is similar to rundown, evidence of short-term depression when the first spike in a burst is larger than mid-burst spikes, traditionally explained by a depletion model (Liley and North, 1953
). Note this type of response for 0 and 1 hr/day chronic stimulation, but the 3 hr/day condition had the same amplitudes for first spike and mid-burst. This can partially explain the larger amplitudes with chronic stimulation, but again, within the resolution of our recordings, overlapping spikes and neurons with different identities are involved.
3.7. Compound Action Potentials from Overlapping Spikes
Even when action potentials appear non-overlapping and distinct, overlap is still possible. shows an example where two spikes appear in response to the 1st pulse with 12 and 14 ms delay (Ai), whereas a single, larger spike appears in response to the 2nd pulse (Aii). Using computer addition of the first two smaller spikes at variable offsets shows that they can be matched very well to the later large spike (6 μV rms error) (Aiii). This strongly suggests that the change in apparent amplitude is due to serendipitous alignment of action potentials from two neurons detected at one electrode. shows an analogous case with two closely spaced spikes in response to the 1st pulse (Bi) but only one for the 2nd pulse (Bii). Superposition of waveforms (not shown) was unsatisfactory (rms error > 10 μV), suggesting that the disappearing waveform was due to failure of the neuron to fire at 13 ms rather than overlap. While selective testing of the compound action potential-overlap hypothesis can be convincing, systematically quantifying the contribution of overlap to the overall amplitude change was judged too cumbersome due to the thousands of possible spike combinations.
The 2nd pulse evokes responses with larger spike amplitudes due to the recruitment of different units and compound action potentials
3.8. Most examples of short-term spike amplitude plasticity arise from spikes of differing latencies
We examined whether spike responses that occurred at the same time could arise from the same unit with a possible plastic gain in amplitude in response to the 2nd stimulus possibly enhanced by chronic stimulation. In order to search for short-term spike amplitude plasticity as a possible explanation for differences in amplitude seen with chronic stimulation, we further separated the records into those where the first spikes had nearly the same latency in response to the 1st and 2nd pulses (differences < 0.5 ms). These comprised 45–50% of spikes. shows that there were no significant changes in amplitude for spikes of the same latency, regardless of chronic stimulation or distance from the stimulus. The changes in amplitude were confined to the population of spikes with different latencies which were positively affected by chronic stimulation, consistent with . For 0 and 1 hr/day chronic stimulation, the slopes were similar with a 0.8 μV change in amplitude per μm distance, while the 3 hr/day condition evoked a 60% higher slope of 1.3 μV/μm. This suggests that the amplitude effect was caused by different identities and numbers of units being recorded with different latencies rather than spike amplitude plasticity of the same unit with the same latency.
The 2nd pulse does not induce spike amplitude plasticity, independent of the chronic stimulation and distance from the stimulus
3.9. Pulse order effects on evoked spikes
We found no difference in the number of responses evoked by the 1st or 2nd pulses. However, there were notable differences in the statistics for records in which there were evoked responses to both pulses (50%) or to only one of the pulses (50%). Figure 11Ai-vi illustrates three cases we distinguished. shows that, when both pulses were effective, more spikes were elicited (2.5 spikes/stimulus) than when only one of the pulses elicited spikes (1.5 spikes/stimulus). The 3 hr/day chronic stimulation further increased these rates. In addition, shows that spike amplitudes were 40–80% higher for responses to both pulses of the paired stimulus compared to responses to only one of the stimuli.
When 1st & 2nd pulses both evoke a response, more spikes with higher amplitudes are seen than when a pulse evokes only one response