| Budget Amount *help |
¥3,340,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥240,000)
Fiscal Year 2007: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2006: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Background : Propofol has an aspect of addictive agent. Neuronal mechanisms of addiction are thought to be related with the activation of midbrain dopamine system originated from ventral tegmental area (VTA). In this study, we planed to characterize propofol effects for the excitability of VTA dopaminergic neurons using patch clamp technique.
Method : Brain slices containing VTA were obtained from young rats. Membrane potentials and membrane currents of the VTA neurons were recorded using whole-cell patch clamp technique. We obtained data from the neurons exhibiting voltage sag under current clamp mode in response to negative current injection and time-ependent inward current under voltage clamp mode in response to hyperpolarizing steps, which is characteristic to midbrain dopaminergic neurons. Propofol was diluted into artificial cerebrospinal fluid (ACSF) to a final concentration of 0.5 to 50μM and bath-applied for 6 to 16min until responses were stabilized. Membrane potential and act
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ion potentials were recorded under current clamp mode and membrane currents at a holding potential of -60mV were recorded under voltage clamp mode in the different setting. Same experiments were done using THIP (4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridine-3-ol, 3 and 30μM), a selective GAGAA agonist. In some experiments of voltage clamp mode, ACSF was supplemented with 100□M of picrotoxin for blocking GABAA receptor.
Result : Action potentials were increased with 0.5 and 5μM of propofol perfusion in 8 of 18 neurons under current clamp configuration (control : 9.3±4.2 spikes/min, 0.5μM:27.5±0.1 spikes/min, 5μM:19.0±8.2 spikes/min, washout : 1.8±0.8 spikes/min, p<0.05). In other ten neurons, action potentials were unchanged or suppressed (control: 0.75±0.71 spikes/min, 0.5μM:0.43±0.37 spikes/min, 5μM:0.06±0.04 spikes/min, washout : almost no spikes, p>0.05). Neurons activated low-ose propofol showed shallower membrane potential (propofol activated neurons: -53.4±4.7 mV, propofol insensitive neurons : -55.5±4.0mV, p<0.05), and more intrinsic excitability compared to propofol insensitive or depressed neurons. With THIP perfusion, action potentials were increased in two of four neurons at 3μM although action potential was completely suppressed at 30μM. In the voltage clamp experiment, 0.5 and 5μM of propofol induced inward currents, which represents cation influx or anion exflux, in 6 of 11 neurons (inward current group : control : 0.35±0.18pA/pF, 0.5μM:-0.76±0.20 pA/pF, 5μM:-0.93±0.20 pA/pF, washout : 0.48±0.51pA/pF, n=6, outward current group : control : -0.36±0.11 pA/pF, 0.5μM: -0.31±0.14pA/pF, 5pM:0.15±0.24pA/pF, washout : 0.73±0.46pA/pF, n=5). THIP 3μM could not induce significant inward current for seven neurons and 30μM induced marked outward current. Inward currents induced by low-ose propofol completely suppressed when 100□M of picrotoxin was added in ACSF.
Conclusion : Low-ose propofol can activate some population of VTA domaminergic neurons, which has shallower membrane potential and more intrinsic excitability than the propofol-insensitive or suppressed neurons. THIP, selective GABAA agonist, partially mimicked the propofol effect and picrotoxin blocked it, this phenomenon would be related to GABAA receptor activation of propofol on VTA neurons. This result can account for previous in vivo study^1.
1. Anesth Analg 2002 ; 95 : 915.9 Less
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