478.03/JJJ17. High-speed visualization of calcium mediated sub-threshold oscillations in the pedunculopontine nucleus using a novel ratiometric imaging technique.

HYDE JR; URBANO FJ; GARCIA-RILL E

San Diego, CA, USA.

Congreso; 2013 Society for Neuroscience Meeting.; 2013

Society for Neuroscience

The pedunculopontine nucleus (PPN) is a component of the reticular activating system, and is involved in the activated states of waking and paradoxical sleep. Gamma oscillations (~20-80 Hz) are evident in all PPN neurons and are mediated by high threshold voltage-dependent N- and P/Q-type calcium channels. We tested the hypothesis that high-speed calcium imaging would reveal calcium-mediated oscillations in synchrony with patch clamp recorded oscillations during depolarizing current ramps. This study also attempts to characterize cellular sub-domains using a novel ratiometric technique using Oregon Green BAPTA-1 (OGB1) with coinjections of a new long-stokes-shift dye, Chromeo 494 (CHR). Electrical responses were recorded using whole cell patch clamp electrodes using 9-16 day old sagittal rat brain slices. Slices were recorded at 37°C perfused with oxygenated aCSF in an immersion chamber containing the synaptic blockers gabazine (GABAA antagonist), strychnine (glycine antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (AMPA/kainate receptor antagonist), and APV (NMDA receptor antagonist), and also Tetrodotoxin to block sodium channels. Recording electrodes also contained Fura-2, Bis Fura-2, or OGB1/CHR calcium sensitive dye (n=121, dye filled n=67, control n=54). Calcium transients were visualized using a high speed, dual camera imaging system (Evolve 128, Photometrics) and xenon lamp or LED illumination. Cells manifested calcium transients with oscillations in both somatic and proximal dendrite fluorescence recordings. Fluorescent calcium transients were blocked with the nonspecific calcium channel blocker, cadmium, or the combination of ω-Agatoxin-IVA (AgA), a specific P/Q-type calcium channel blocker, and ω-conotoxin-GVIA (CgTx), a specific N-type calcium channel blocker. Overlaying calcium and electrical recordings showed highly coincident oscillation peaks. Preliminary data suggests the presence of distributed oscillatory sub-domains. Each domain oscillated with a local phase and timing while the oscillations seen in electrical recordings in the soma may be an average of the activity in sub-domains. This study showed that high-speed calcium imaging is a viable method for measuring sub-threshold calcium oscillations with high spatial and temporal resolution in cellular sub-domains. The OGB1/CHR ratiometric indicator proved comparable to traditional calcium indicators for accuracy while allowing high-speed image acquisition. GRANTS: NIH award R01 NS020246; NIH award P20 GM103425; FONCyT, Agencia Nacional de Promoción Científica y Tecnológica; BID 1728 OC.AR. PICT 2008-2019.