Neurosteroids modulate epileptiform activity and associated high-frequency oscillations in the piriform cortex

Abstract

Allotetrahydrodeoxycorticosterone (THDOC) belongs to a class of pregnane neurosteroidal compounds that enhance brain inhibition by interacting directly with GABAA signaling, mainly through an increase in tonic inhibitory current. Here, we addressed the role of THDOC in the modulation of interictal- and ictal-like activity and associated high-frequency oscillations (HFOs, 80-500 Hz; ripples: 80-200 Hz, fast ripples: 250-500 Hz) recorded in vitro in the rat piriform cortex, a highly excitable brain structure that is implicated in seizure generation and maintenance. We found that THDOC: (i) increased the duration of interictal discharges in the anterior piriform cortex while decreasing ictal discharge duration in both anterior and posterior piriform cortices; (ii) reduced the occurrence of HFOs associated to both interictal and ictal discharges; and (iii) prolonged the duration of 4-aminopyridine-induced, glutamatergic independent synchronous field potentials that are known to mainly result from the activation of GABAA receptors. Our results indicate that THDOC can modulate epileptiform synchronization in the piriform cortex presumably by potentiating GABAA receptor-mediated signaling. This evidence supports the view that neurosteroids regulate neuronal excitability and thus control the occurrence of seizures.

Keywords: 4-aminopyridine; 4AP; ACSF; HFOs; THDOC; allotetrahydrodeoxycorticosterone; artificial cerebrospinal fluid; epileptiform synchronization; high-frequency oscillations; ictogenesis.

Fig. 1

Interictal and ictal discharges in the piriform cortex. (A) Epileptiform discharges recorded from the anterior and posterior piriform cortices of an intact brain slice following the application of 4AP. The spectrograms show the initial negative shift followed by oscillations in the 10–20 Hz range characterizing the ictal event. The ictal onset and an interictal discharge are expanded in insets a and b, respectively. (B) Time delay histograms between the anterior and posterior regions calculated for interictal and ictal discharges. These are pooled data from 19 slices. Note the jittering in the region of onset, as no event initiated in one region in particular. Events in the anterior piriform cortex are used as the reference (time 0). (C) Epileptiform activity does not change over time during 4AP application (n=5 slices). Upper panels show the duration and interval of occurrence of interictal events over time, respectively while lower panels show the duration and interval of occurrence of ictal events; data were averaged in epoques lasting 5 min. Time 0 represents the time of appearance of spontaneous epileptiform activity.

Fig. 2

Modulation of Interictal discharges by THDOC. (A) Representative examples of interictal events in the 4AP control, 4AP+0.1 μM THDOC and the 4AP +5 μM THDOC condition. (B) Bar graph showing the average duration of 4AP-induced slow events. THDOC induced a significant increase in duration of these slow events in the anterior region. (C) Bar graph showing the change in interval of interictal events normalized to the 4AP control condition (100%). THDOC induced a significant increase compared to 4AP. (D) Change in amplitude of interictal events, note that THDOC induced a significant increase of amplitude compared to 4AP. (*p<0.05; 4AP pooled data from n=13 slices, 0.1 μM THDOC n=7 slices, 5 μM THDOC n=7 slices).

Fig. 3

Effect of THDOC on Ictal-like events. (A) Representative examples of ictal discharges in the 4AP control condition, 4AP+0.1 μM THDOC and the 4AP +5 μM THDOC condition. (B) Bar graph showing the average duration of ictal discharges following the application of 4AP and THDOC. THDOC induced a significant decrease in ictal discharge duration compared to 4AP in both the anterior and posterior regions of the piriform cortex. (C) Number of ictal discharges per hour following in the 4AP control condition, 4AP+0.1 μM THDOC and 4AP+0.5 μM. The frequency of ictal discharges did not change significantly following the application of THDOC. (*p<0.05 4AP pooled data n=13, 0.1 μM THDOC n=7, 5 μM THDOC n=7).

Fig. 4

HFOs during interictal discharges. (A) Example of an interictal discharge co-occurring with a ripple (a) or with a fast ripple (b) in the anterior piriform cortex under 4AP bath application. Note in b that the fast ripple is visible on the descending phase of the spike. (B) Bar graph showing the proportion of interictal events, in the anterior and posterior piriform cortex, co-occurring with HFOs in the 4AP control (anterior: n=19, events=1235; posterior: n=19, events=1047), 4AP+0.1 μM THDOC (anterior: n=7, events=665; posterior: n=7, events=659) and the 4AP+5 μM THDOC (anterior: n=6, events=633; posterior: n=6, events=636) condition. The application of THDOC induced a significant decrease in the proportion of interictal spikes co-occurring with HFOs, mainly in the fast ripple frequency range and in the anterior region. (*p<0.05) Note that the proportion of interictal spikes co-occurring with HFOs in the posterior region of the piriform cortex is almost negligible.

Fig. 5

HFOs during ictal discharges. (A) Representative 4AP-induced ictal discharge from the anterior and posterior piriform cortex, with filtered traces showing HFOs (red circles) in the ripple and fast ripple frequency ranges. (B) Rates of ripples and fast ripples over time during 4AP induced ictal discharges in the anterior and posterior regions (data pooled from n=19 slices). Note that ripples and fast ripple rates are significantly higher in the anterior compared to the posterior region. During the pre- and post-ictal periods, rates of fast ripples are also significantly higher in the anterior region of the piriform cortex compared to the posterior region. (C) Bar graphs showing the average number of HFOs per ictal discharge following the application of 4AP (n=13), 0.1 μM (n=7 slices) and 5 μM THDOC (n=6 slices). Application of THDOC induced a significant decrease in the occurrence of HFOs in the anterior region, but not in the posterior region. (D) Line graph showing the change in ictal duration compared to the change in HFO rate following the application of either 0.1 or 5 μM THDOC. All events are normalized to the 4AP control condition. (*p<0.05, **p<0.01, ***p<0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Effects of THDOC on slow events (A). Recording of 4AP control activity, followed by the application of CPP and NBQX, 0.1 μM THDOC and then 5 μM THDOC in the anterior and posterior piriform cortex. Insets a and b show an example of a slow event on an expanded time scale. (B) Bar graphs showing the change in duration of these slow spikes in the 4AP+0.1 μM (n=4 slices) and 4AP+5 μM (n=4 slices) THDOC. Duration is expressed as the change from the 4AP control condition. (*p<0.05).