IFIBYNE   05513
INSTITUTO DE FISIOLOGIA, BIOLOGIA MOLECULAR Y NEUROCIENCIAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
478.05/JJJ19. Visualizing P/Q- and N-type calcium channels in the pedunculopontine nucleus.
Autor/es:
LUSTER BR; HYDE JR; GARCIA-RILL E; URBANO FJ
Lugar:
San Diego, CA, USA.
Reunión:
Congreso; 2013 Society for Neuroscience Meeting.; 2013
Institución organizadora:
Society for Neuroscience
Resumen:
The pedunculopontine nucleus (PPN) is involved in the activated states of waking and paradoxical sleep, forming part of the reticular activating system. Every PPN cell fires maximally at gamma band activity (~30-60Hz). Previous patch clamp studies showed that this oscillatory activity is due mainly to P/Q- type and partially N-type voltage-gated calcium channels. The purpose of this study was to confirm that the wide field fluorescent calcium signal in the cell as a whole is mainly due to P/Q-type channels and only partially due to N-type calcium channels. These studies also compared two custom LED illumination systems. 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 Oregon Green BAPTA 1 (OGB1). Calcium transients were visualized using a wide field high sensitivity electron-multiplying camera (Evolve 512, Photometrics). Images of the whole cell from eight ramps were averaged to produce the final calcium image series for each cell. Four illumination systems were compared in the 480nm range: a 300W xenon lamp, 100W halogen lamp, simple ultra-high brightness LED (Luminus Devices), and a stabilized LED (LED with a photodiode feedback loop). Cells (n=24) generated calcium signals in both the soma and dendrites. Fluorescent calcium signals were completely blocked with a combination of 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. Calcium signals were mostly blocked when AgA was used by itself. This study demonstrated that the fluorescent calcium signal of the cells is mainly due to P/Q-type voltage-gated calcium channels and only minimally due to N-type voltage-gated calcium channels. This was made possible by the use of a stable, low noise LED illumination system and high sensitivity camera. GRANTS: NIH award R01 NS020246; NIH award P20 GM103425; FONCyT, Agencia Nacional de Promoción Científica y Tecnológica; BID 1728 OC.AR. PICT 2008-2019.