Interneuronal Network Activity at the Onset of Seizure-Like Events in Entorhinal Cortex Slices

The number of inter-ictal IPSCs (black bar) versus inter-ictal EPSCs (gray bar) and of SLEs preceded by either outward (black bar) or inward PSCs (gray bar) recorded in 15 experiments are shown in the left and right bar histograms, respectively.

Double cell recordings during 4-AP-induced SLEs. A, Schematic drawing of the experimental configuration, showing the patch pipettes onto a FS-IN and principal PyrN located in mEC layer V and the double-barreled pipette used to record [K⁺]_o signals. Rf, Rhinal fissure LII, Layer II; LV, Layer V. B, Representative current-clamp and voltage-clamp recordings from a FS-IN (middle trace) and a PyrN (lower trace) and simultaneous [K⁺]_o shift (top trace) during a 4-AP-induced ictal discharge. Insets, The typical firing of the patched neurons. Expansion of the traces outlined by the box in B is shown in the bottom. C, Vertical dotted lines mark the correlation between neuronal firing and the changes in K⁺. D, Representative double current-clamp recordings from a FS-IN and a PyrN and simultaneous shift in [K⁺]_o (top trace) during a 4-AP-induced ictal discharge. Expansion of the traces outlined by the box in D is illustrated in E (bottom). Inset calibration bars: 500 ms, 500 pA.

Effects of NMDA/AMPA receptor antagonists on 4-AP-induced SLEs. A, Representative voltage-clamp recording from a PyrN (bottom trace of the pair) and simultaneous changes in [K⁺]_o (top trace) during a 4-AP-induced SLE characterized by interneuronal-network onset. The ii-IPSC in the box is enlarged in the bottom pair of traces. B, Effect of NMDA/AMPA receptor antagonists coperfused with 4-AP (gray shaded bar) on both PyrNs activity and the associated [K⁺]_o changes. The traces outlined by the boxes are expanded in the lower part of the panel. C, Box plot representation of the increase in [K⁺]_o during 4-AP-(left) and 4-AP+NBQX/APV (right) treatments. Delta [K⁺]_o represents absolute value additional to the simultaneous correspondent [K⁺]₀. The box ranges indicate the 25th and 75th percentile and whiskers represent SD values. Significance at Mann–Whitney test: **p < 0.01.

Correlation between IPSCs and [K⁺]_o. A, Typical 4-AP-evoked ii-IPSC recorded from a mEC PyrN (bottom trace) and the associated [K⁺]_o signal (top trace). The gray-shaded areas represent the ii-IPSC current charge and the relative integral of cumulative K⁺ charge at its peak value. B, Plot of the areas shaded in gray computed for different ii-IPSPs. Each symbol refers to a different PyrN. Regression lines corresponded to correlation coefficients (R²) values between 0.709 and 0.989. C, Shaded areas outline K⁺ and IPSC charge values calculated as in A for pre-ictal events. D, Relationship between the area of 4-AP-induced π-IPSCs and the relative cumulative K⁺ charge. High correlation coefficients (R²) included between 0.7 and 0. 9 were observed in all neurons.

[K⁺]_o changes associated to different 4-AP-induced epileptiform events. A, Box plots comparing the increase in [K⁺]_o associated with ii-IPSC, π-IPSC, ii-EPSC, and π-EPSC) and [SLEs preceded by IPSCs (“inhibitory” onset SLE) or EPSCs (excitatory onset SLE). The box ranges indicate the 25th and 75th percentile and whiskers represent SD values. Significance at Mann–Whitney test: **p < 0.001 and ***p < 0.0001; p = 0.3, 0.6, 0.09; NS, No significance). Significance at two-sample Student t test: *p < 0.05. B, Scar plot showing the amount of K⁺ released in the extracellular space during 4-AP-evoked ii-IPSCs (black dots) and π-IPSCs (gray dots). K⁺ changes induced by π-IPSCs are larger than those induced by ii-IPSCs. C, Bar histograms of the [K⁺]_o slope value (left) and the time required to reach the maximum peak of the K⁺ traces (right) associated to the ii-IPSC (black columns) and pi-IPSC (gray columns) events. Data are normalized to the mean value of inter-ictal events. Significance at paired-sample Student t test: *p < 0.05, **p < 0.001. D, Scar plot of the cumulate K⁺ charges during 4-AP-evoked ii-IPSCs (black square dots) and a pi-IPSCs (gray round dots). K⁺ changes induced by pi-IPSCs larger and more sustained in time.