Abstracts

Rationale: We have previously demonstrated that illumination of a low concentration of caged GABA with a UV LED can release sufficient GABA to attenuate seizure-like activity in both cultured hippocampal neurons and brain slices. In a new set of experiments, we have explored the anticonvulsant potential of a novel caged GABA, ruthenium-bipyridine-triphenylphosphine-GABA (RuBi-GABA), which releases GABA when illuminated by blue light. Methods: We used standard techniques to prepare 600 μm cortical brain slices, which were perfused with artificial cerebrospinal fluid (ACSF) in a chamber with a glass coverslip forming the bottom. A blue light LED (emission maxima of 470 nm) package was placed below the coverslip allowing unfocused light to directly illuminate the slice from below. A computer generated TTL pulse controlled the LED. In our initial experiments we illuminated the slice during perfusion with ACSF and monitored the temperature with a small thermocouple as a function of time and LED current. We then elicited seizure-like discharges by perfusion with ACSF lacking magnesium and containing 100 μM 4-aminopyridine (0 Mg/4-AP ACSF) and examined the effect of 4.5 sec light illumination (200 mA) in the absence and presence of RuBi-GABA (3 or 10 μM). When we varied current through the LED, the highest temperature increases observed during a 30 sec observation period were 0.18 ± 0.09, and 0.19 ± 0.08°C, at 200, and 400 mA, respectively. Our subsequent RuBi-GABA uncaging experiments then utilized 200 mA of current for 5 sec. Results: To test whether RuBi-GABA uncaging had an effect on epileptiform events, we compared the number of paroxysmal bursts, number of spikes, and baseline standard deviation (SD) in the 4.5 sec epoch before illumination and 4.5 sec epoch during illumination. When the slice was perfused with 0 Mg/4-AP ACSF lacking RuBi-GABA, LED illumination had no effect on the burst number, spike number, or baseline SD (p>0.06 for all 3 situations). In the presence of 10 μM RuBi-GABA, however, burst number decreased from 1.167± 0.219 to 0.145 ± 0.177; spike number diminished from 3.437 ± 1.003 to 0.382 ± 0.307 and baseline SD was reduced from 0.063 ± 0.015 to 0.032 ± 0.007 mV (p<0.001 for all 3 parameters). When the RuBi-GABA concentration was reduced to 3 μM, burst number; spike number, and baseline SD were also reduced but the reduction did not reach statistical significance (p>0.05 for all groups). Conclusions: Our results indicate that micromolar RuBi-GABA can release sufficient GABA when illuminated with a blue light to suppress paroxysmal activity in a 600 μm thick rat brain slice. As the RuBi-GABA is activated with visible light, it can provide greater tissue penetration and less photo-toxicity than previously used UV-sensitive caged compounds. In addition, ruthenium-based uncaging has faster photorelease kinetics. If future rodent testing verifies the efficacy of RuBi-GABA for in vivo focal seizure models without demonstrable toxicity, caged compounds will become an appropriate target for more intense therapeutic investigation.