Optical suppression of seizure-like activity with an LED

Abstract

The therapy of focal epilepsy remains unsatisfactory for as many as 25% of patients. We tested the hypothesis that an efficient, ultraviolet light emitting diode (UV LED), coupled with a newly developed "caged" gamma-aminobutyric acid (GABA), might be capable of terminating "ictal-like" events in cultured murine neurons. GABA was released from BC204, a recently described caged GABA, using a small, ultraviolet (UV) LED. Ictal-like events were provoked by removal of extracellular magnesium. In preliminary control experiments, the concentration of GABA released from our caged compound was dependent upon the strength and duration of the illumination, and readily achieved micromolar (microM) levels that are known to activate tonic, extrasynaptic GABA(A) receptors. Ultraviolet illumination had no effect when BC204 was not present in the perfusate and the currents produced by BC204 were eliminated by picrotoxin. Within a few seconds of UV illumination, BC204 rapidly terminated ictal-like events at low microM concentration. Uncaging of BC204 also blocked the elevation of intracellular calcium induced by seizure-like discharges in our cultures. While much more technical development is clearly required to extend our observations to a more intact preparation, these results suggest the intriguing possibility of constructing an implantable device to "optically suppress" focal human seizures under closed loop control.

Figure 1

Duration response relationship for BC204 activation with a UV LED. A. Increasing the duration of illumination from 1 to 6 sec lengthens the duration and magnitude of inward current. B. Average peak ± 1 SD current responses for illumination durations up to 15 sec, showing that beyond 6 sec, the response reaches a plateau (n= 4 cells). Values were normalized to 4 sec light application.

Figure 2

Concentration and current response relationships for GABA and BC204. A. Concentration response curve for GABA between 1 and 10 μM, normalized to the 10 μM response (n = 4–7 cells for each point). B. Increasing the current driving the UV LED (50 – 250 mA) causes an increase in inward current produced by BC204 (30 μM) uncaging. C. Inward, GABAergic current plotted as function of UV LED current and normalized to current produced by 10 μM GABA (n = 4 – 13 cells for each point). Data for both 10 and 30 μM BC204 are plotted. For 30 μM BC204, the current produced by LED current ≥ 150 mA exceeds the current produced by 10 μM GABA. D. Combining the data in A and B allow the GABA concentration produced by BC204 illumination to be estimated. LED currents below 100 mA produced GABA currents below the bottom of our dose response curve when we used 10 μM BC204. The abscissa is the same in C and D.

Figure 3

Examples of spontaneous activity and seizure-like events in two neurons under different experimental conditions. A1. Under control conditions (extracellular calcium and magnesium = 2 and 1 mM, respectively and no BC204), there were often some spontaneous postsynaptic potentials and action potentials. A 4 sec illumination with the UV LED (hv) had no effect on resting potential. A2. In the presence of BC204 (30 μM) and magnesium-free perfusate, UV illumination (200 mA for 4 sec) rapidly terminates the action potentials and quiets the voltage trace. A3. Lower power illumination (25 mA) has no effect on spiking or the baseline. B1. Under control conditions, another neuron shows some spontaneous bursting. B2. In the same cell, removing extracellular magnesium led to an abrupt, “seizure-like” increase in action potential firing that was unaffected by UV (200 mA) in absence of BC204. Scale in A1 applies to all traces. Arrows in A1 and B1 point to artifacts generated by the dish heater cycling.

Figure 4

Quantification of seizure-like behavior in cultures. A. The number of spikes in a 10 sec interval was correlated with the standard deviation (mV) of the recording baseline (156 intervals; r² = 0.66). B. In control and zero magnesium extracellular perfusate, UV LED illumination does not alter excitability in the absence of BC204 (^{a, b} p = 0.25 and 0.46, respectively, compared to control plus light, n = 12–24 cells; ^c,d p = 0.39 and 0.10, respectively, compared to zero magnesium plus light, n = 6–17 cells, both by Student-Newman-Keuls test (SNK)). The larger baseline standard deviation and spike counts in the illuminated zero magnesium cultures reflects the variability in the seizure-like activity produced by magnesium withdrawal. Baseline standard deviations and spike numbers were significantly different between control and zero magnesium groups (p < 0.001 and p < 0.05, respectively by SNK). C1. In the presence of BC204 (30 μM) and zero magnesium, UV LED illumination using 200, 150, or 100 mA of current significantly reduced baseline standard deviation and spike number compared to 10 sec epochs in which there was no UV LED illumination (^a p < 0.001 by SNK, compared to BC204 “no light”; ^b p < 0.05 by SNK, compared to BC204 “no light”, n = 9 – 11 cells). C2. In the presence of BC204 (10 μM) and zero magnesium, illumination with 200, 150, or 100 mA of UV LED current again significantly reduced both parameters compared epochs in which there was no UV LED illumination (^a p < 0.001 by SNK, compared to “no light”; ^b p < 0.05 by SNK, compared to “no light”, n = 6 cells). C1 and C2 have the same abscissa. Bars in A –C are SEM.

Figure 5

Effect of UV LED illumination on ionized intracellular calcium levels in neocortical cultures. Both traces show calcium peaks in normal extracellular magnesium, likely representing spontaneous bursting. Each figure shows averages of all neurons in a field (16 and 14, respectively for A and B) and then a single neuron, showing that the calcium elevations are synchronous A. Intracellular calcium levels rapidly rise and then oscillate when magnesium is removed from the extracellular solution. Three separate applications of UV LED illumination (hν = 200 mA for 5 sec) fail to alter the oscillation of the calcium levels. B. Intracellular calcium levels begin to rise and oscillate at the time of magnesium removal and BC204 addition. However, two light applications (hv) dramatically reduced ionized calcium. In both A and B, there are gaps in the trace coinciding with illumination, because the UV LED emission spectra overlaps the filters used for Fluo-3.