Optical triggered seizures using a caged 4-Aminopyridine

Abstract

Animal models of epilepsy are critical not only for understanding the fundamental mechanism of epilepsy but also for testing the efficacy of new antiepileptic drugs and novel therapeutic interventions. Photorelease of caged molecules is widely used in biological research to control pharmacologic events with high spatio-temporal resolution. We developed a technique for in vivo optical triggering of neocortical seizures using a novel caged compound based on ruthenium photochemistry (RuBi-4AP). Epileptiform events in mouse cortex were induced with blue light in both whole brain and focal illumination. Multi-electrode array recording and optical techniques were used to characterize the propagation of these epileptic events, including interictal spikes, polyspikes, and ictal discharges. These results demonstrate a novel optically-triggered seizure model, with high spatio-temporal control, that could have widespread application in the investigation of ictal onset, propagation and to develop novel light-based therapeutic interventions.

Keywords: caged compound; electrophysiology; epilepsy model; neocortex; optical imaging; photostimulation.

Figure 1

Optical triggering of epilepsy using topical application of RuBi-4-AP in vivo. (A) Schematic diagram of experimental design. RuBi-4-AP (0.1 ml, 15 mM) is topically applied to induce seizures. RuBi-4-AP is stable in ACSF solution (Left) and the 4-AP and ruthenium complex are photoreleased with visible 470 nm blue light (Right). The black arrow on the image of the cortical surface indicates the LFP electrode. (B) 5 min baseline LFP recording shows normal brain activity after RuBi-4-AP application before uncaging. (C) Interictal discharges and polyspikes are induced by 10 s blue light illumination (470 nm, 1.2 A) over the neocortex. The duration of illumination is shown with a blue line (Top). (D) Ictal discharge is induced by 300 s blue light pulses. The expanded view (Bottom) shows a typical ictal discharge starting with an initial spike (ictal onset). (E) Ictal-like activity is also induced by longer, 600 s, illumination. Ictal discharges are recorded during illumination and even after the blue light is turned off (Top). The middle (1) and bottom panels (2) show expanded views of two ictal discharges in the upper panel.

Figure 2

Optical triggering of focal epilepsy using focal illumination and RuBi-4-AP in vivo. (A)RuBi-4-AP (2 μl, 25 mM) is injected into the neocortex to induce seizures. RuBi-4-AP is photoreleased with an optical fiber (470 nm LED). The white arrow on the image of cortical surface indicates the LFP electrode and the white bar shows the optical fiber. Left: green light; Middle: blue light; and Right: merged image. (B) Baseline recording shows normal brain activity after RuBi-4-AP application before uncaging. (C) LFP recording (black) with light on marker. Interictal discharges and polyspikes are induced by 1 s focal blue light. The blue bar shows the LED light-on period. (D) One ictal discharge is induced by 60 s blue light illumination (Top). The further expended view (Bottom) shows an ictal-like discharge induced by focal blue light illumination.

Figure 3

Multi-contact laminar depth array recording of in vivo RuBi-4-AP epileptic events. The depth electrode is placed 0.2 mm away from the site of focal illumination. (A) Focal photostimulation 0.2 mm away from the laminar depth electrode placed tangentially into the cortex. (B) A 1 s pulsed photostimulation (blue line) results in a short duration event that occurred simultaneously in all layers. (C,D) Longer duration illumination (60 s) results in immediate onset of polyspikes and short duration events involving all layers simultaneously. (E) Occasionally, layer specific events (black arrow) occur that quickly spread to all other layers. (F) However, the majority of events involve all layers simultaneously. The blue bars in (B,C) show the timing of blue photostimulation.

Figure 4

Optical mapping of in vivo ictal discharge. (A,B) 530 nm map and 780 nm map of a focal ictal discharge induced by RuBi-4AP and photostimulation. The left panel shows an image of the cortical surface and images at selected time points with respect to seizure onset (0 s). The right panel shows time course of the LFP (local field potential), CBV (cerebral blood volume), and 780 nm signals recorded during one seizure. Red dots in the images are the location of the ROIs in the seizure focus. (C,D) 530 nm map and 780 nm map of a focal ictal discharge induced by 4-AP. The left panel shows an image of the cortical surface and images at selected time points with respect to the seizure onset. The right panel shows the time course of LFP, CBV, and 780 nm signals during one seizure. Red dots in the images are the locations of the ROIs in the seizure focus.

Figure 5

CBV and light scattering response to ictal discharges elicited by RuBi-4-AP or 4-AP. (A) Average seizure duration induced by RuBi-4-AP is significantly shorter compared to seizure duration induced by 4-AP (p < 0.05). (B) Average total LFP power of seizures induced by RuBi-4-AP was significantly decreased compared with the total LFP power of seizures induced by 4-AP (p < 0.01) (C) Average maximum response of CBV and 780 nm signals in the seizure focus was significantly decreased in the RuBi-4-AP compared with the 4-AP seizures (CBV: P = 0.04; 780 nm: p = 0.023). ^*p < 0.05; ^**p < 0.01.