Optical control of focal epilepsy in vivo with caged γ-aminobutyric acid

Abstract

Objective: There is enormous clinical potential in exploiting the spatial and temporal resolution of optical techniques to modulate pathophysiological neuronal activity, especially intractable focal epilepsy. We have recently utilized a new ruthenium-based caged compound, ruthenium-bipyridine-triphenylphosphine-γ-aminobutyric acid (RuBi-GABA), which releases GABA when exposed to blue light, to rapidly terminate paroxysmal activity in vitro and in vivo.

Methods: The convulsant 4-aminopyridine was used to induce interictal activity and seizures in rat neocortical slices and anesthetized rats. We examined the effect of blue light, generated by a small, light-emitting diode (LED), on the frequency and duration of ictal activity in the presence and absence of RuBi-GABA.

Results: Neither blue light alone, nor low concentrations of RuBi-GABA, affected interictal activity or baseline electrical activity in neocortical slices. However, brief, blue illumination of RuBi-GABA, using our LED, dramatically reduced extracellular spikes and bursts. More impressively, illumination of locally applied RuBi-GABA rapidly terminated in vivo seizures induced by topical application of 4-aminopyridine. The RuBi-GABA effect was blocked by the GABA(A) antagonist picrotoxin, but not duplicated by direct application of GABA.

Interpretation: This is the first example of optical control of in vivo epilepsy, proving that there is sufficient cortical light penetration from an LED and diffusion of caged GABA to quickly terminate intense focal seizures. We are aware that many obstacles need to be overcome before this technique can be translated to patients, but at the moment, this represents a feasible method for harnessing optical techniques to fabricate an implantable device for the therapy of neocortical epilepsy.

FIGURE 1

Effect of ruthenium-bipyridine-triphenylphosphine–γ-aminobutyric acid (RuBi-GABA) on interictal bursts in a brain slice. (A) Interictal bursts induced by 4-aminopyridine were not affected by illumination in the absence of RuBi-GABA. A2 shows the illuminated portion of A1 on an expanded scale. (B) When 10µm RuBi-GABA was present, illumination (light-emitting diode [LED] current 200mA) suppressed individual spikes and bursts. B2 shows the illuminated portion of B1 on an expanded scale. The spike and burst suppression greatly outlasts the slice illumination in B because of GABA persistence after uncaging. (C) After the addition of picrotoxin (100µm) to the perfusion fluid, illumination of RuBi-GABA no longer prevented spikes and bursts. C2 shows the illuminated portion of C1 on an expanded scale. The horizontal bars in A, B, and C represent 5 seconds of LED illumination.

FIGURE 2

Summary of ruthenium-bipyridine-triphenylphosphine–γ-aminobutyric acid (RG) effect on (A) spikes, (B) interictal bursts, and (C) baseline standard deviation (SD). The black bars represent the values of the parameters in the 4.5 seconds immediately preceding illumination, and the gray bars represent the values of the same parameters in the 4.5 seconds during illumination, allowing a 0.5-second window for illumination electrical artifact. Illumination (light-emitting diode [LED] current 200mA) had no effect on any of the 3 parameters in the absence of RG. However, in the presence of 10µm RG, 5 seconds of illumination (current 200mA) significantly reduced all 3 parameters (p < 0.01, paired t test). When the LED current was reduced to 100mA, the spikes and bursts were still diminished (p < 0.01, paired t test), but baseline SD was not significantly reduced (p > 0.05). At 3µM RuBi-GABA, spikes, bursts, and baseline SD were reduced, but not significantly (p > 0.05). In the presence of 10µm RG plus picrotoxin (PTX; 100µm), 5 seconds of illumination no longer reduced any of the 3 parameters (p > 0.05, paired t test or Wilcoxin signed rank) (a, p > 0.05; b, p < 0.01).

FIGURE 3

Effect of blue light-emitting diode (LED) illumination on slice and brain surface temperature. (A) Upper plot shows the maximum temperature change found in a brain slices during a 30-second period of blue LED illumination (n = 8 measurements at each current). The lower plot shows the maximum temperature change during the 5-second illumination period (n = 8 measurements at each current power level). A 400mA LED current raised slice temperature <0.2°C after 30 seconds. (B) The top line represents the maximum temperature increase recorded from the cortical surface after 60 seconds of blue light illumination (n = 6 measurements at each current). The bottom line shows the maximum temperature change after 30 seconds of light flash.

FIGURE 4

Examples of 4-aminopyridine (4-AP)-induced neocortical seizures and effect of ruthenium-bipyridine-triphenylphosphine–γ-aminobutyric acid (RuBi-GABA). (A) Control seizure induced by 4-AP lasted 140 seconds. (B) Blue light illumination (500mA) had no effect on seizure duration in the absence of RuBi-GABA. (C) Without illumination, local administration of RuBi-GABA (5µM) did not shorten seizure durations. (D) The seizure duration significantly diminished with pairing of the light flash (500mA) and RuBi-GABA (5µM). The horizontal bar represents 60 seconds of blue light-emitting diode illumination (B and D).

FIGURE 5

Photorelease of γ-aminobutyric acid (GABA) from ruthenium-bipyridine-triphenylphosphine–GABA (RG) significantly reduces the duration of neocortical seizures. For each animal, the seizure durations before, during, and after blue light illumination were compared. Seizure duration was not affected by illumination in the absence of RG (a, p > 0.4, 1-way repeated measures analysis of variance [ANOVA] on ranks). However, illumination (30 or 60 seconds) significantly reduced the duration of seizures in the presence of different concentrations of RG (b, c, p < 0.05 and p < 0.001, respectively, compared to preillumination and postillumination durations; 1-way repeated measures ANOVA followed by Student-Newman-Keuls; n = 7 or 8 animals in each group). Picrotoxin (PTX; 100µm) eliminated the effect of RG illumination (n = 4 animals). Focal dural reservoir application of GABA at seizure onset also had no effect on seizure durations (n = 8 animals). There was no significant difference in seizure durations before and after illumination across all the groups, indicating no effect of RuBi-GABA on seizure durations in the absence of illumination (p > 0.4 by ANOVA). The shaded bars for focal GABA administration represent before, during, and after GABA application, not illumination.

FIGURE 6

Rapid seizure termination after illumination of ruthenium-bipyridine-triphenylphosphine–γ-aminobutyric acid (GABA) is blocked by picrotoxin (PTX) and not duplicated by local GABA application. (A) In the presence of PTX, illumination of caged GABA for 60 seconds (bar) no longer stopped seizures. (B) When GABA was quickly applied into the dural reservoir (short, lower bar), the seizures were also unaffected. (C) A longer electroencephalographic recording shows 2 control seizures, followed by a third seizure that was unaffected by rapid application of GABA (arrow). The duration of a fourth seizure that came 3 minutes later was shorter than the 3 prior seizures, suggesting that GABA application eventually had an effect. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]