| Budget Amount *help |
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Evaluation of released dopamine amount within brain slices by inhibition of [^<11>C] raclopride binding using a dynamic autoradiography technique.
To elucidate the mechanism of in vivo binding competition between radioligand and endogenously released transmitter, we examined the influence of depolarization-induced dopamine (DA) release on [^<11>C] raclopride (RAC)-specific binding to D_2 receptors in slices of living brain tissues using dynamic position autoradiography. Rat brain slices were incubated in a chamber with [^<11>C]RAC in oxygenated Krebs-Ringer solution. Two-dimensional images of radioactivity in the slices were recorded on a Storage Phosphor Screen and dynamic changes were measured every ten minutes. [^<11>C]RAC took 2 hours to reach a steady-state in the brain slices. When the brain slices were exposed to the depolarization agents (25 mM K^+, 50 mM K^+ and 20 μM veratridine), striatal [^<11>C]RAC-specific binding decreased to 91%, 56% and 46% of the control, respectively.
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However, pre-exposure of brain slices to the same depolarization treatment (50 mM K^+) for 30 min did not affect the [^<11>C]RAC-specific binding suggesting that the reduction in receptor density and/or affinity was not involved in the decrease of [^<11>]RAC-specific binding. The [^<11>C]RAC-specific binding to striatum decreased dose-dependently in the presence of DA with an IC_<50> value of 45 μM.The synaptic DA concentration during each depolarization treatment estimated from the acquired does-response curve, agreed proportionally with the amount of DA released into the Krebs-Ringer solution by the treatment. These results demonstrated that the competition of [^<11>C]RAC with endogenous DA can be involved, rather than changes of D_2 receptor density and/or affinity, in the decrease of [^<11>C]RAC-specific binding in living brain slices following evoked DA release, and possibly, in living brain observed in vivo with PET as well.
A new approach to quantify the dynamic changes of acetylcholine synthesis and release in living brain slices by dynamic autoradiography technique.
We have developed a new autoradiographic (ARG) method to estimate the dynamic changes of acetylcholine synthesis and release in living brain slices by using [^<11>C]choline. Brain slices were incubated with [^<11>C]choline in oxygenated Krebs-Ringer medium and serial two-dimensional time-resolved images of [^<11>C]choline uptake and release of redioactivity were obtained on the Storage Phosphor screens. The [^<11>C]choline uptake was increased with period of incubation and the more [^<11>C]choline uptake was found in striatum than cerebral cortex. Pretreatment of K^+ depolarization-induced release of endogenous transmitter enhanced the [^<11>C]choline uptake in striatum but did not in cerebral cortex. The [^<11>C]choline uptake in striatum was significantly diminished by Hemicholinium-3(HC-3), an inhibitor of high-affinity choline uptake. Uptake of [^<11>C]choline in brain slices was saturable and two different uptake systems, high- and low-affinity choline uptake, were confirmed by kinetic analysis using the Lineweaver-Burk plots. The accumulated ^<11>C radioactivity in the striatum was released by the treatment of Veratridine (depolarization agent) and HC-3. This pattern od disappearance was good accord with that of appearance of acetylcholine in the medium. These results indicate that this method is useful to obtain the information about acetylcholine synthesis and release in living brain slices. Less
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