Elevation of potassium concentration transforms nonsynaptic ictal-like activity into status epilepticus–like activity
About 20 min after the onset of perfusion with aCSF containing EGTA, 5 mM, and K+, 6 or 7.5 mM, over the exposed hippocampus, synaptic transmission was suppressed in the CA1 region, and a type of ictal-like slow-wave activity appeared with (n = 7/12) or without (n = 5/12) superimposed spikes. This slow-wave activity was similar to the nonsynaptic epileptiform activity both in vitro (16) and in vivo (19). Large-amplitude spontaneous spikes recorded from the pyramidal layer represent the simultaneous firing of a population of neurons. To distinguish between spontaneous spikes and evoked population spikes (PSs), “spike” is used to indicate spontaneous population spikes. Figure 1 shows an example of slow-wave activity with superimposed burst spikes. During baseline recording (Fig. 1A), orthodromic Schaffer collateral stimulation evoked large field PSs in the CA1 pyramidal layer and field excitatory postsynaptic potentials (fEPSPs) in the CA1 stratum radiatum. No epileptiform activity was observed in the spontaneous potentials. Thirty minutes after the application of EGTA, 5 mM, and K+, 7.5 mM, slow-waves superimposed by burst spikes appeared with suppressed evoked potentials, indicating a decrease of synaptic transmission (Fig. 1B). Increasing K+ to 15 mM changed the slow-waves into persistent double-spike activity repeated at a frequency of ∼4 Hz lasting >30 min (Fig. 1C). The slow waves with superimposed spikes returned when K+ in the aCSF solution was changed back to 7.5 mM (Fig. 1D). EGTA, 5 mM, was applied through the whole period. The suppression of synaptic transmission indicated by a decrease of the evoked responses was persistent during the switches of epileptic patterns caused by the changes of K+ concentrations (Fig. 1B–D).
Figure 1. High K+ transformed slow waves with spike superimposed into continuous double spikes. A: Baseline recording with small potential in the pyramidal layer (Pyr.) and stratum radiatum (S. rad.) in the spontaneous potentials, and with large population spikes (PSs) and field excitatory postsynaptic potentials (fEPSPs) in the evoked potentials. B: After 30-min application of artificial CSF with K+, 7.5 mM, and EGTA, 5 mM, slow-waves superimposed by spikes appeared in the spontaneous potentials with potential shifts negative in the pyramidal layer and positive in the stratum radiatum. Synaptic transmission was mostly blocked, indicated by PS disappearing with a small fEPSP in the evoked potentials. C: After perfusion with K+, 15 mM, continuous double spikes repeated at a frequency of ∼4 Hz replaced slow waves. D: Slow waves with spikes reappeared when K+ returned to 7.5 mM.
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The application of aCSF with EGTA, 5 mM, and K+, 6 or 7.5 mM, sometimes induced slow-waves without superimposed spikes (Fig. 2). Baseline recordings in Fig. 2A show normal evoked responses and normal spontaneous potentials in both the pyramidal layer and the stratum radiatum. Thirty-five minute application of a solution containing EGTA, 5 mM, and K+, 7.5 mM, induced low-frequency high-amplitude slow waves without superimposed spikes in the spontaneous potentials. This low-frequency activity occurred when the evoked fEPSP decreased significantly and the evoked PS disappeared, thereby indicating suppression of synaptic transmission (Fig. 2B). The absence of spontaneous spikes in slow-waves did not necessarily mean absence of firing. It could also be the absence of phase-locked firing. After the elevation of K+ in the solution to 15 mM, persistent double-spike activity replaced the slow waves with a frequency of 3 to 4 Hz and large amplitude in the pyramidal layer (Fig. 2C). The spiking activity did not stop until the K+ concentration was returned to 7.5 mM, and the lower frequency slow-waves reappeared (Fig. 2D).
Figure 2. High K+ changed slow-wave activity without superimposed spike into continuous spiking activity. A: Baseline recording with small potential in the pyramidal layer (Pyr.) and theta activity in the stratum radiatum (S. rad.) in spontaneous potentials, and with large population spikes (PSs) and field excitatory postsynaptic potentials (fEPSPs) in the evoked potentials. B: After 35-min application of artificial CSF with K+, 7.5 mM, and EGTA, 5 mM, low-frequency high-amplitude slow waves appeared in the spontaneous potentials while synaptic transmission was blocked, indicated by PSs disappearing and small fEPSPs in the evoked potentials. C: After perfusion with elevated K+ to 15 mM, slow waves were changed into double-spike bursts with a frequency of 3 to 4 Hz. D: Slow waves reappeared when K+ returned to 7.5 mM.
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These results indicate that the slow-wave burst pattern induced by EGTA, 5 mM, and K+, 6 or 7.5 mM, was transformed into persistently recurring double population spikes when K+ was increased to 12 or 15 mM. Similar nonsynaptic persistent spike activity could also be generated directly from a nonepileptic condition. As shown in Fig. 3, the application of K+, 12 mM, with Ca2+, 2 mM, could not induce epileptiform activity despite an increase in neuronal excitability indicated by multiple-PSs in evoked potentials (Fig. 3A). Even the removal of Ca2+ from the perfusion solution with K+, 12 mM, did not induce epileptiform activity in the spontaneous potentials, although the evoked PSs were larger in the pyramidal layer (Fig. 3B). However, continuous spikes appeared almost immediately after the addition of EGTA, 5 mM, in the solution and lasted >1 h. At the beginning, the spike activity was observed as separate spikes with higher frequencies and higher amplitudes (Fig. 3C and E). About 20 min later, the activity appeared as paired spikes repeated at a frequency of ∼4 Hz (Fig. 3D and E, diamond line) while synaptic transmission was suppressed, as indicated by the disappearance of PSs and very low amplitude fEPSPs in the evoked potentials.
Figure 3. Status epileptiform activity induced by K+, 12 mM, and EGTA, 5 mM. A–D: Spontaneous potentials (left) and orthodromic evoked potentials (right) in the CA1 pyramidal layer and stratum radiatum. A: After 35-min application of artificial CSF with K+, 12 mM, and Ca2+, 2 mM, no epileptiform activity was observed in spontaneous potentials despite the increase of neuronal excitability indicated by multiple population spikes (PSs) in evoked potentials. B: After 35-min application of K+, 12 mM, without Ca2+, still no epileptiform activity was observed in spontaneous potentials, although larger multiple PSs were evoked in the pyramidal layer. C: Spikes appeared in the spontaneous potentials after the application of EGTA, 5 mM, to reduce the [Ca2+]o further and partially suppress the synaptic transmission. D: Persistent double spikes appeared in the spontaneous potentials after 25 min of EGTA application, and the synaptic transmission was mostly blocked, indicated by the disappearance of PSs and very small field excitatory postsynaptic potentials in evoked potentials. E: Spikes appeared ∼5 min after the application of EGTA, 5 mM, and lasted >1 h. During the early 20 min, the activity was observed as separated spikes with higher frequencies and higher amplitudes than those of double spikes that appeared later when the synaptic transmission was suppressed. The frequency of double spikes indicated by the diamond line in the left panel was half of the single-spike frequency.
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These data suggest that the 12 mM K+ concentration with normal Ca2+ concentration applied to the exposed surface of hippocampus cannot evoke epileptiform activity, whereas the combination of low Ca2+ concentration and elevated K+ concentration can induce sustained nonsynaptic double-spike activity that is different from low-Ca2+ slow-wave activity. The amplitude, frequency, and duration of both the nonsynaptic slow-wave activity and the double-spike activity are compared in Table 1. The duration of a single double-spike event (0.038 ± 0.039 s) was significantly shorter than that of a slow-wave event (1.24 ± 0.31 s; n = 12; p < 0.01). The frequency of the double spike (3.77 ± 1.28 Hz) was significantly higher than that of the slow wave (0.34 ± 0.14 Hz; n = 12; p < 0.01).
Table 1. Comparison among nonsynaptic slow waves, nonsynaptic double-spike activity, and PTX-induced interictal and ictal activity
|Activity||n||Frequency (Hz)||Duration (s)||Amplitude (mV) Pyramidal layer||Amplitude (mV) Stratum radiatum|
|Slow waves||12||0.34 ± 0.14|| 1.24 ± 0.31||1.20 ± 0.48|| 3.25 ± 1.10|
|Double spikes||12|| 3.77 ± 1.28a|| 0.038 ± 0.039a||1.78 ± 1.21|| 1.00 ± 0.37a|
|PTX: interictal|| 5|| 0.28 ± 0.07b|| 0.14 ± 0.01b||2.19 ± 0.71|| 4.11 ± 1.34|
|PTX: ictal|| 5|| 0.09 ± 0.03 c|| 0.69 ± 0.21 c||6.79 ± 1.36|| 9.57 ± 1.79|
The nonsynaptic continuous double spike repeated at a frequency of ∼4 Hz was quite different from the traditional synaptic epileptiform activity induced by convulsants, usually with long silent intervals between bursts. It has been shown that elevating K+ concentration can increase the frequency of burst activity (2,20–22) because of a decrease in the potassium AHP current. In addition, reducing Ca2+ concentration can also decrease inhibitory activity (15). Therefore we hypothesized that a decrease in inhibitory activity, such as GABAergic inhibition and AHP activity, was the underlying mechanism generating the high frequency of continuous double-spike activity observed in this investigation. This hypothesis was investigated by measuring multiple unit activity (MUA) and by comparing these waveforms with picrotoxin (PTX)-induced synaptic epileptiform activity.
Decrease of inhibitory activity by high-concentration K+ and low-concentration Ca2+
To study the gradual changes of neural activity, the exposed hippocampus was perfused first with an aCSF solution containing a 12 mM concentration of K+ for 40 min, and then with a zero-Ca2+ aCSF for another 40 min, and finally, EGTA (5 mM) was added.
During baseline recording with a perfusion solution of K+, 5 mM, and Ca2+, 2 mM, paired-pulse stimulation with an interval of 25 ms evoked a large PS in the first response (PS1) but no PS in the second response (PS2), indicating strong GABAergic inhibition (e.g., paired-pulse inhibition, PPI) in the CA1 region. After paired-pulse stimulation, a long inhibition period (IP) with a low level of MUA activity indicated a long period of AHP with limited neural activity (Fig. 4A). Elevating K+ to 12 mM in the solution changed the single PS1 into multiple PSs and decreased paired-pulse inhibition, as indicated by the appearance of a second population spike (PS2; Fig. 4B). Although the IP duration did not change significantly in the MUA recording, the appearance of an afterdischarge within the IP immediately after the paired-pulse stimulation (open arrow in Fig 4B) indicates an increase in neural excitability by high K+. After the removal of Ca2+ in the high-K+ perfusion solution, the amplitude of PS2 increased, and IP duration decreased significantly, indicating a further decrease of both GABAergic inhibition and AHP (Fig. 4C). The EGTA solution induced single spontaneous spike activity, reduced synaptic transmission, as indicated by a smaller PS1 amplitude with increased PS1 latency, and significantly decreased PPI and IP duration (Fig. 4D). Thirty minutes after the application of EGTA, spontaneous double-spike activity continued, and synaptic transmission was mostly blocked (Fig. 4E) with small IP duration.
Figure 4. Change of the inhibition period (IP) after spikes during the process of [K+]o increase and [Ca2+]o decrease. A–D: Right: Multiple unit activity (MUA) signal in the CA1 pyramidal layer obtained by a high-pass filter with a cutoff frequency of 500 Hz. Solid arrow, Artifacts and responses of paired-pulse stimulation in the Schaffer collaterals. After the stimulation, a significant IP was apparent with little neural activity. The MUA potentials are truncated in the range from −0.05 mV to +0.05 mV. Left: Expanded evoked responses of paired-pulse stimulation with 25-ms interval indicate the recurrent inhibition (paired-pulse inhibition; PPI). A: During baseline recording, IP was long, and no second PS (PS2) was observed in the paired-pulse response, indicating strong after-hyperpolarization (AHP). B: High K+, 12 mM, decreased the PPI, as indicated by the appearance of PS2 in the evoked potential, and reduced the AHP, as indicated by the appearance of an afterdischarge in MUA after the stimulation (open arrow). C: After the removal of Ca2+ in the perfusion solution, PPI and IP decreased further. D: Ten minutes after the EGTA solution, spontaneous spike activity appeared, and PPI and IP decreased significantly. E: Thirty minutes after EGTA solution, evoked PSs disappeared, and double spikes continued in the spontaneous potential of the pyramidal layer (see expansion on the right).
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The mean IP duration in the final 20 min with 12 mM K+, zero Ca2+ (0.52 ± 0.08 s) was significantly shorter than that in the final 20 min with 2 mM Ca2+ (1.14 ± 0.44 s; F= 13.9; p = 0.003; n = 8; repeated measures ANOVA). The mean IP duration in the first 20 min of EGTA application was even shorter (0.30 ± 0.09 s). Because the amplitudes of both first and second evoked PS in the paired-pulse response were larger during the periods of K+ increase and Ca2+ reduction (Fig. 4B–D) than during the baseline recording (Fig. 4A), the decrease of IP could not be caused by a depression of evoked potentials. Therefore the decrease of IP could indicate a significant decrease of AHP duration by low-Ca2+ concentration. In contrast, IP durations after bursts induced by GABAA-receptor blockader PTX were significantly longer.
It has been shown in vitro that a PTX-induced epileptiform burst was followed by a long AHP because of a calcium-dependent potassium potential in hippocampal neurons (23–25). A similar phenomenon was also observed in this preparation. Application of PTX, 0.4 mM, with K+, 7.5 mM, over the surface of the exposed hippocampus, first, induced interictal bursts followed 30 min later by both interictal and ictal bursts (Fig. 5). Interictal activity in the CA1 pyramidal layer was characterized by a short positive wave superimposed with negative population spikes and followed by a long silent interval. The duration of the inhibition period with low MUA activity after the interictal bursts was 0.8 to 4.5 s (Fig. 5A). Ictal activity was characterized by a longer burst made up of several interictal bursts. A long IP duration (2.5–9.0 s) followed a long ictal burst (Fig. 5B). The frequencies of both PTX-induced interictal (0.28 ± 0.07 Hz) and ictal (0.09 ± 0.03 Hz) bursts were significantly lower than the frequency of EGTA-induced nonsynaptic double-spike activity (3.77 ± 1.28 Hz; p < 0.001; Table 1).
Figure 5. Inhibition period (IP) after interictal and ictal activity induced by picrotoxin (PTX). A, B: first row, Spontaneous epileptiform activity in the CA1 pyramidal layer; second row, corresponding Multiple unit activity (MUA) signal in the CA1 pyramidal layer obtained by a high-pass filter with a cutoff frequency of 500 Hz and truncated in the range from −0.05 mV to +0.05 mV. The application of PTX, 0.4 mM, with K+, 7.5 mM, first induced interictal activity, succeeded 30 min later by both interictal and ictal activity. A: IP durations in MUA signal after interictal activity were 0.8 to 1.5 s. B: IP duration after ictal activity was longer (8.9 s).
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These data indicate that the decrease of GABAergic inhibition by PTX alone can induce bursts with long AHP inhibition intervals, thereby reducing the burst frequency. In contrast, high [K+]o combined with low [Ca2+]o can decrease both the GABAergic inhibition and the AHP inhibition, thereby shortening the inhibition duration after neural firing activity and producing the high burst frequency.