Abstracts

Caged compounds are inert prodrugs that are capable of releasing small bioactive molecules when excited by light at the appropriate wavelength. They were initially developed to facilitate rapid, step increases in the local concentration of neurotransmitters and are typically activated with a very brief pulse from a mercury bulb or laser. We wondered whether we could use low levels of light from an ultraviolet diode (UV LED) to release GABA from a new caged analog and modulate seizure-like activity in cultured neurons., We used cultured hippocampal neurons after about 2 weeks [italic]in vitro [/italic]and recorded in whole cell bridge or voltage clamp mode. GABA was applied by whole bath perfusion or by uncaging 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy)carbonyl]amino]butanoic acid (BC204). A UV LED (Nicchia 365 nm/100 mW) was activated by a custom fabricated driver for 4 seconds in the uncaging experiments., We first verified that our UV LED was capable of generating sufficiently high GABA concentrations to activate GABA[sub]A [/sub]receptors in our cultures. We found that driver currents between 25 and 250 mA produced readily detectable currents in the presence of BC204 (30 [mu]M). These currents are well below the maximum capacity of the UV LED (700 mA). The current elicited by 150 mA corresponded to a 10 [mu]M GABA current, when normalized to a GABA dose response curve. The currents produced by uncaging BC204 were reduced by 89% in the presence of picrotoxin (100 [mu]M). BC204 itself at 30 [mu]M is a very weak GABA[sub]A[/sub] blocker, reducing 3 [mu]M GABA currents by about 5%. When cultures were exposed to medium lacking magnesium, they generated increased synaptic activity, paroxysmal depolarization shifts, and often [quot]seizure like[quot] discharges that were abolished by perfusion with GABA as low as 3 [mu]M. In the presence of BC204 (30 [mu]M), a 4 second illumination with 100-200 mA also eliminated spontaneous activity. The figure below shows the effect of a 4 second light pulse on bursting.[figure1]Illumination in the absence of BC204 produced no detectable current., These results suggest that it may be possible to utilize a compact, low power UV LED in combination with locally applied caged GABA to activate tonic GABA[sub]A[/sub] and GABA[sub]B[/sub] receptors and modulate paroxysmal activity. These receptors can be activated by GABA concentrations in the low micromolar range, which it may be possible to achieve [italic]in vivo[/italic]., (Supported by Alafi Family Foundation, the NIH (R01 NS 42936 to SMR), and NSF (MCB-8920118 to BFS).)