Persistent ictal-like activity in rat entorhinal/perirhinal cortex following washout of 4-aminopyridine

Abstract

Application of 4-aminopyridine (4-AP, 100μM) in a solution containing 0.6mM Mg(2+) and 1.2mM Ca(2+) to hippocampal-entorhinal-perirhinal slices of adult rat brain induced ictal-like epileptiform activity in entorhinal and perirhinal cortices as revealed by electrophysiological field potential recordings. The ictal-like activity persisted after washing out the 4-AP. This persistence indicated that a change had occurred in the slice so that it was now "epileptic" in the absence of the convulsant 4-AP. Induction of persistent ictal-like activity was dependent upon the concentration of divalent cations during 4-AP exposure; that is, although 4-AP caused ictal-like activity in approximately half the slices in solution containing 1.6mM Mg(2+) and 2.0mM Ca(2+), this ictal-like activity did not persist upon washout of the 4-AP. Expression of the persistent ictal-like epileptiform activity required ionotropic glutamate-mediated synaptic transmission: application of the AMPA/kainate receptor antagonist NBQX after 4-AP washout reduced persistent ictal-like activity, and the combined application of NBQX and the NMDA receptor antagonist d-AP5 completely blocked it. In order to investigate the mechanism of induction of persistent ictal-like activity, several agents were applied before the introduction of 4-AP. Application of d-AP5 did not block the onset of ictal-like activity upon introduction of 4-AP but did prevent the persistence of the ictal-like activity upon washout of the 4-AP. In contrast, induction of persistent ictal-like activity was not prevented by simultaneous application of the group I metabotropic glutamate receptor (mGluR) antagonists LY 367385 and MPEP or by application of the protein synthesis inhibitor cycloheximide. In conclusion, we have characterized a new in vitro model of epileptogenesis in which induction of ictal-like activity is dependent upon NMDA receptor activation but not upon group I mGluR activation or protein synthesis.

Fig. 1. Ictal-like and/or status epilepticus-like events could be recorded in entorhinal and perirhinal cortices after application of 4-aminopyridine

Slices bathed in 0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution. A: Ictal-like event illustrating tonic and clonic components. Field electrode in entorhinal cortex layer II/III. B: SE-like event in a different slice. Full event lasted over 29 min. Field electrode in perirhinal cortex layer II/III. a: Trace filtered after digitization using a 0.05 Hz high-pass RC filter. b: Same SE-like event on an expanded time scale. Trace in b not filtered after digitization.

Fig. 2. Application of 4-AP in a solution containing 1.6 mM Mg²⁺ and 2.0 mM Ca²⁺ induced ictal-like epileptiform activity in entorhinal cortex which did not persist after washing out the 4-AP

4-AP (100 μM) caused ictal-like activity in the entorhinal cortex in 1.6 mM Mg²⁺/2.0 mM Ca²⁺ solution in 10 of 22 slices; however, the ictal-like activity did not persist upon washout of the 4-AP (n = 5 of 5). A: In this slice the ictal-like activity was replaced with interictal-like activity at approximately 12 min of 4-AP washout. Trace filtered after digitization using a 0.5 Hz high-pass RC filter. B: Expanded traces from early (a) and late (b) in the washout showing an ictal-like event from point a in A and interictal-like events from point b in A. Traces in B were not filtered after digitization. C: Summary of 5 slices which had ictal-like activity in 4-AP, comparing percent time spent in ictal-like activity before and after 4-AP washout (Wilcoxon).

Fig. 3. Application of 4-AP in a solution containing 0.6 mM Mg²⁺/1.2 mM Ca²⁺ induced ictal-like epileptiform activity in entorhinal/perirhinal cortex which persisted after washing out the 4-AP

A: Bathing slice in 0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution did not cause epileptiform activity in this slice (left). Addition of 4-AP (100 μM) caused ictal-like epileptiform activity (middle). Ictal-like epileptiform activity persisted upon washout of 4-AP (right, same trace as Fig. 1A). Right trace taken 42 min after initiation of 4-AP washout. All traces in A recorded from same slice. Field electrode in A in entorhinal cortex layer II/III. B: A different slice showing persistence of ictal-like activity over 57 min of 4-AP washout. Field electrode in B in perirhinal cortex layer II/III. C: Scatter plot of control group B slices showing length of longest epileptiform event in each slice in 4-AP and after 4-AP washout. N=30 in 4-AP and n=32 after 4-AP washout. The two triangles denote the two slices in which the longest event after 4-AP washout was measured but the longest event in 4-AP was not measured due to a 4-AP exposure < 40 min. D: Bar graph of control group B slices comparing % time spent in SE + ictal-like events before and after 4-AP (Wilcoxon, n= 30).

Fig. 4. Raising divalent cation concentration to 1.6 mM Mg²⁺/2.0 mM Ca²⁺ after complete 4-AP washout reduced but usually did not block ictal-like activity

A: Recording from entorhinal cortex in 0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution in 4-AP (top), after 4-AP washout for 32 min (middle) and after 31 min in 1.6 mM Mg²⁺/2.0 mM Ca²⁺ solution (bottom). B: % time spent in SE + ictal-like events after 4-AP washout fell upon raising the divalent cation concentration (n = 8, measured for the 10 min period immediately prior to initiation of the solution change and for the 10 min period between 30 and 40 min following initiation of the solution change). Triangles represent the slice in A. C: Longest epileptiform event in 4-AP washout in each slice before and after raising the divalent cation concentration. Three of eight slices had no ictal-length events after raising divalent cation concentration. The unpaired point represents the slice that had only SE-like activity before the divalent cation concentration was raised. Excluding unpaired point, P = 0.237, n = 7 (Wilcoxon).

Fig. 5. Expression of persistent ictal-like activity after 4-AP washout was dependent upon ionotropic glutamatergic synaptic transmission

A: Persistent, ictal-like epileptiform activity after 4-AP washout (top, full ictal-like event lasted 185 s) was reduced but still present after application of the NMDA receptor antagonist D-AP5 (middle), but all epileptiform activity was blocked after subsequent application of the AMPA/kainate antagonist NBQX (bottom). Data in A recorded in perirhinal cortex (PRC) in the same slice at 56 min of 4-AP washout (top), at 58 min of D-AP5 exposure (middle), and at 17 min of NBQX exposure (bottom). B: Persistent, ictal-like epileptiform activity after 4-AP washout (top, full ictal-like event lasted 168 s) was reduced but still present after application of the AMPA/kainate antagonist NBQX (bottom) in this slice. Data in B recorded in PRC in the same slice at 58 min of 4-AP washout (top) and at 37 min of NBQX exposure (bottom). C: Percent time spent in SE + ictal-like events in entorhinal cortex (EC) in each slice fell with addition of D-AP5 (n=6; measured for the last 10 min of 4-AP wash before the D-AP5 application and for the 10 minutes between 20 and 30 minutes of D-AP5 application). D: Percent time spent in SE + ictal-like events in EC in each slice fell with addition of NBQX (n=9; measured for the last 10 min of 4-AP wash before the NBQX application and for the 10 minutes between 20 and 30 minutes of NBQX application). E: Bar graph illustrating % of slices still showing ictal-like activity after D-AP5, after NBQX, and after both D-AP5 and NBQX in EC and PRC. F: Bar graph illustrating % time spent in ictal-like events after D-AP5, after NBQX, and after both D-AP5 and NBQX. (There was no SE-like activity.) Percent time spent in ictal-like events was significantly lower in the presence of only NBQX than in the presence of only D-AP5. Protocol was 4-AP, then 4-AP washout, then application of ionotropic glutamate antagonist, then sometimes application of second ionotropic glutamate antagonist. Slice bathed in 0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution throughout protocol.

Fig. 6. NMDA receptor antagonist did not block the onset of ictal-like activity but did block the induction of persistent ictal-like activity

A: Summary graph showing % time spent in ictal-like events in each successive condition in each slice in EC. (There was no SE-like activity.) Protocol of experiment shown at top. Each slice represented by a different symbol. Pre-incubation with the NMDA receptor antagonist D-AP5 in 1.6 mM Mg²⁺/2.0 mM Ca²⁺ solution (a), did not prevent the onset of ictal-like activity in 4-AP/0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution (b), but did prevent persistence after 4-AP washout (c). B: Traces from EC in same slice (represented by square in A) in each condition. Lower case letters match letters on x-axis in A. Middle trace (b) was recorded in the presence of D-AP5 at 58 min of 4-AP application. C: Ictal-like epileptiform activity stopped within 20 min of 4-AP washout in continued presence of D-AP5 (recording continued for an additional 11 min. not shown). Simultaneous recording from EC and PRC from same slice as B. Traces filtered after digitization using a 0.3 Hz high-pass RC filter. D: Bar graph comparing D-AP5 slices to control group C slices showing a smaller percent time spent in ictal-like events during 4-AP exposure for D-AP5 slices in EC but no significant difference between D-AP5 slices and control slices in PRC (n=18 control, n= 4 D-AP5; NS = not significant compared to either of the other two bars). E: Bar graph comparing control group C slices to D-AP5 slices showing persistent ictal-like activity after 4-AP washout in control slices but none in D-AP5 slices, either in EC or PRC (n=18 control, n= 4 D-AP5).

Fig. 7. Group I metabotropic glutamate receptor antagonists did not prevent the induction of persistent ictal-like epileptiform activity

A: Summary graph showing percent time spent in ictal-like events in each successive condition in each slice in EC. Protocol of experiment shown at top. Each slice represented by a different symbol. Pre-incubation with the group I mGluR antagonists LY 367385 (LY) and MPEP in 1.6 mM Mg²⁺/2.0 mM Ca²⁺ solution for 40 min (a), did not prevent the onset of ictal-like epileptiform activity upon application of 4-AP in 0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution (b), and the ictal-like epileptiform activity persisted after washing out 4-AP (c). B: Traces from EC in same slice (represented by circle in A) in each condition. Lower case letters match letters on x-axis in A. Top trace (a) recorded in 1.6 mM Mg²⁺/2.0 mM Ca²⁺ solution in the presence of LY/MPEP. Middle trace (b) recorded at 36 min of 4-AP application in the presence of LY/MPEP. Bottom trace (c) recorded at 53 min of 4-AP washout in the continued presence of LY/MPEP. C: Scatter plot comparing control group B slices and LY/MPEP slices showing length of longest epileptiform event in each slice after 4-AP washout. D: Bar graph shows no difference between control group B and LY/MPEP slices in percent time spent in SE + ictal-like events after 4-AP washout (n = 32 control and n = 4 LY/MPEP).

Fig. 8. The protein synthesis inhibitor cycloheximide did not prevent the induction of persistent ictal-like epileptiform activity

A: Pre-incubation of slices in cycloheximide for 30 min in 0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution before introduction of 4-AP did not prevent the onset of ictal-like activity in 4-AP (top) and did not prevent the persistence of ictal-like epileptiform activity upon washout of 4-AP (bottom). Top trace recorded in EC at 34 min of 4-AP exposure. Bottom trace recorded in EC in same slice at 48 min of 4-AP washout. B: Scatter plot comparing control group A slices and cycloheximide slices showing length of longest epileptiform event in each slice after 4-AP washout. C: Bar graph shows no difference between control group A slices and cycloheximide slices in percent time spent in SE + ictal-like events after 4-AP washout (n = 18 control and n = 6 cycloheximide). All slices bathed in 0.6 mM Mg²⁺/1.2 mM Ca²⁺ solution throughout time in recording chamber.