Initiation and slow propagation of epileptiform activity from ventral to dorsal medial entorhinal cortex is constrained by an inhibitory gradient

Abstract

Key points: The medial entorhinal cortex (mEC) has an important role in initiation and propagation of seizure activity. Several anatomical relationships exist in neurophysiological properties of mEC neurons; however, in the context of hyperexcitability, previous studies often considered it as a homogeneous structure. Using multi-site extracellular recording techniques, ictal-like activity was observed along the dorso-ventral axis of the mEC in vitro in response to various ictogenic stimuli. This originated predominantly from ventral areas, spreading to dorsal mEC with a surprisingly slow velocity. Modulation of inhibitory tone was capable of changing the slope of ictal initiation, suggesting seizure propagation behaviours are highly dependent on levels of GABAergic function in this region. A distinct disinhibition model also showed, in the absence of inhibition, a prevalence for interictal-like initiation in ventral mEC, reflecting the intrinsic differences in mEC neurons. These findings suggest the ventral mEC is more prone to hyperexcitable discharge than the dorsal mEC, which may be relevant under pathological conditions.

Abstract: The medial entorhinal cortex (mEC) has an important role in the generation and propagation of seizure activity. The organization of the mEC is such that a number of dorso-ventral relationships exist in neurophysiological properties of neurons. These range from intrinsic and synaptic properties to density of inhibitory connectivity. We examined the influence of these gradients on generation and propagation of epileptiform activity in the mEC. Using a 16-shank silicon probe array to record along the dorso-ventral axis of the mEC in vitro, we found 4-aminopyridine application produces ictal-like activity originating predominantly in ventral areas. This activity spreads to dorsal mEC at a surprisingly slow velocity (138 μm s^-1 ), while cross-site interictal-like activity appeared relatively synchronous. We propose that ictal propagation is constrained by differential levels of GABAergic control since increasing (diazepam) or decreasing (Ro19-4603) GABA_A receptor activation, respectively, reduced or increased the slope of ictal initiation. The observation that ictal activity is predominately generated in ventral mEC was replicated using a separate 0-Mg²⁺ model of epileptiform activity in vitro. By using a distinct disinhibition model (co-application of kainate and picrotoxin) we show that additional physiological features (for example intrinsic properties of mEC neurons) still produce a prevalence for interictal-like initiation in ventral mEC. These findings suggest that the ventral mEC is more likely to initiate hyperexcitable discharges than the dorsal mEC, and that seizure propagation is highly dependent on levels of GABAergic expression across the mEC.

Keywords: dorsal-Ventral gradient; entorhinal cortex; hyperexcitability.

Figure 1. 4‐AP induced ictal‐ and interictal‐like activity in mEC slices

A, recording position of 16‐shank electrode array on parasagittal mEC slice, with scale depicting dorsal (D), ventral (V), rostral (R) and caudal (C) directions. B, example of ictal‐like bursting activity from dorsal (top) to ventral (bottom) mEC showing burst recorded first in most ventral electrode site (scale bar: 200 μV, 10 s) (a), with zoomed examples of interictal‐like (b) and ictal‐like (c) events (scale bars: 100 μV, 0.5 s and 200 μV, 2 s respectively).

Figure 2. 4‐AP‐induced ictal‐like activity in mEC is initiated in ventral recording sites

A, example recording from a ventrally positioned electrode illustrating an ictal‐like burst (scale bar: 0.2 mV, 2 s) (a), with continuous wavelet transform scalogram illustrating the frequency components of the above recording (b). B, proportion of bursts starting at dorsal and ventral recording sites (n = 123 bursts from 10 slices slices from 8 animals). C, example trace showing 16 channels, filtered in low (10–50 Hz) frequency band (a), with normalized spectral power (b). D, average start time of burst relative to first channel to meet threshold for ictal activity using low frequency filter, showing linear increase with distance from ventral pole; linear regression: R ² = 0.95, P < 0.001, slope = 138 μm s⁻¹. E, example filtered in high (50–250 Hz) frequency band (a), with normalized spectral power (b). F, average burst start time; linear regression: R ² = 0.95, P < 0.001, slope = 157 μm s⁻¹.

Figure 3. Interictal‐like bursts are generated in both dorsal and ventral portions of the mEC

A, an example recording of interictal‐like bursts recorded using a 16‐shank electrode array on parasagittal mEC slice. This 4.5 min segment of data was recorded between 2 ictal‐like bursts (not shown). Numerous interictal‐like bursts were observed, visible on this time scale as brief vertical deflections on the recording. B, individual bursts were detected and clustered into groups according to the time of the waveform peak. In this recording, two groups were identified, the average waveforms of which are depicted in a. b, silhouette plot of the resulting k‐means clustering algorithm. c, the time of the average waveform peak (plotted relative to the time on the most ventral probe) for the two clusters. These data illustrate that interictal bursts are initiated at different points along the dorso‐ventral axis of the mEC. C, probability histograms showing the maximum difference in interictal peak times across all 16 probes for 10 different slices. The mean (μ) maximum difference in peak times is shown for each distribution. These data illustrate that, on average, interictal bursts take 0.2–0.5 s to spread along the dorso‐ventral axis of the mEC.

Figure 4. Intra‐ictal burst waveforms initiated in ventral mEC regions

Aa, example traces from most dorsal (top) and ventral (bottom) recording sites of electrode array (scale bar: 100 μV, 10 s). b, binned cross correlations for every 1 s of data. c, correlation values are shown in the colour axis, with positive peaks indicating ventral‐leading activity and negative peaks dorsal‐leading. B, example of intra‐burst activity across 16‐shank electrode array initiating in ventral mEC during red bar in A (scale bar: 200 μV, 0.5 s). C, lag time associated with peak cross correlation between most ventral sites and each dorsal recording electrode, showing linear increase with distance from ventral pole (linear regression: R ² = 0.93, P < 0.001, slope = 55.9 ± 5 mm s⁻¹). D, proportion of 1 s time bins with correlation peaks in the positive (ventral leading) was greater during ictal events when compared to non‐ictal bins (paired t test, P = 0.002, n = 10 slices from 8 animals). ^*** P < 0.001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5. Ictal‐like bursts show similar propagation in deep cortical layers

A, example silicon probe recording of ictal‐like bursting activity from deep layers (L5/6) of mEC, dorsal (top) to ventral (bottom) (scale bar: 10 z, 20 s), with simultaneous glass electrode recordings from superficial layers (L2/3). B, start time of each recording location relative to initiation of first burst.

Figure 6. Modulation of GABAergic transmission changes rate of ictal‐like propagation in mEC slices

A and B, example traces of ictal‐like events (top) with normalized power (bottom) on 16‐shank recording array after application of 4‐AP (scale bar: 500 μV, 5 s) (Aa and Ba) and subsequent application of diazepam (DZP; 30 μm; Ab) or Ro19‐4603 (RO; 10 nm; Bb). Ac and Bc show time course of ictal burst slope before and after manipulation of GABAergic transmission. C, decreased ictal slope in an example slice after diazepam application (white) compared to 4‐AP alone (grey); 3 ictal bursts shown pre‐ (1800–2400 s) and post‐ (3000–3600 s) drug, with mean slope decreasing ∼2‐fold (b); paired t test, P < 0.001, n = 6 slices from 6 animals. D, ictal propagation is faster after application of Ro19‐4603 (a); paired t test, P < 0.001, n = 6 slices from 6 animals (b). ^*** P < 0.001.

Figure 7. Ictal‐like activity from slices in 0‐Mg²⁺ aCSF is also initiated in ventral mEC recording sites

A, example traces of ictal‐like events (top) with normalized power (bottom) on 16‐shank recording array after Mg²⁺ washout (scale bar: 500 μV, 5 s). B, example burst start times along dorso‐ventral axis of the mEC normalized to start of burst for 3 ictal‐like events recorded after Mg²⁺ washout. C, average slope of ictal initiation under 0‐Mg²⁺ conditions shows bursts occur in the ventral to dorsal direction (55.7 ± 9.4 μm s⁻¹; 1‐way ANOVA, F _1,7 = 8.6, P = 0.02, n = 6 slices from 6 animals). Da, example traces from most dorsal (top) and ventral (bottom) recording sites (scale bar: 100 μV, 10 s); b, 1 s binned cross correlations. c, values shown in colour axis, with positive peaks indicating ventral‐leading activity and negative peaks dorsal‐leading. E, lag time associated with peak cross correlation between most ventral sites and each dorsal recording electrode (linear regression R ² = 0.81, P < 0.001, slope = (35.8 ± 2) × 10³ μm s⁻¹). F, proportion of time bins with correlation peaks in the positive (ventral leading) compared to non‐ictal bins (paired t test, P = 0.06, n = 6 slices from 6 animals). [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 8. Application of 500 nm kainate and 50uM picrotoxin produces interictal‐like events which originate in ventral mEC

A, illustration of relative position of glass recording electrodes in dorsal (top) and ventral (bottom) mEC. B, example trace after application of picrotoxin (50 μm; a); box represents one interictal event (b) with cross correlation (c) showing peak occurring in ventral mEC before dorsal (scale bar: 0.1 mV). C, average time‐pooled data showing the development of burst frequency (min⁻¹) in dorsal (black) and ventral (grey) mEC (n = 8 slices from 5 animals). Continuous line represents mean (± SEM shown by shaded areas). D, mean (±SEM) time until first epileptic event is shorter in ventral than dorsal mEC (paired t test P = 0.013, n = 8 slices from 5 animals). E, average cross correlation between dorsal and ventral events (n = 8 slices from 5 animals) showing peak lag time >0 s. ^* P < 0.05.

Figure 9. Separation of dorsal and ventral mEC produces preferential decrease in epileptic events in the dorsal mEC

A, relative position of dorsal (top) and ventral (bottom) recording electrodes and scalpel cut (dotted line) between electrodes. B, example trace (scale bar 0.2 mV, 30 s). C, averaged time‐pooled data showing the development of burst frequency in dorsal (a) and ventral (b) cut slices compared to control (n = 4). D, zoomed example trace (box) showing desynchronized bursting in dorsal and ventral mEC (scale bar 0.2 mV, 2 s). E, average cross correlation of cut slices (n = 4 slices from 4 animals) compared to controls (n = 8 slices from 5 animals) (a) shows significant decrease in correlation of epileptic bursts (b) (unpaired t test P = 0.031). ^* P < 0.05. F, decreased average burst frequency in dorsal mEC in cut slices compared to control. G, bar graph showing mean (±SEM) time in seconds until first recording epileptic event is also shorter in ventral than dorsal mEC when ends are separated (^*paired t test P = 0.026) (n = 4 slices from 4 animals).