Different approaches to study acetylcholine release: endogenous ACh versus tritium efflux

Summary

Superfused slices of guinea-pig cerebral cortex (CC), caudate nucleus (CN) and thalamus (Th) were used to compare i) the resting and electrically-evoked release of endogenous acetylcholine (ACh) in the presence of physostigmine (Phys) and ii) the resting and electrically-evoked tritium efflux (after preloading with ³H choline) in the absence or in the presence of Phys and hemicholinium (HC-3).

In addition, the effect of GABA, morphine and their antagonists on both effluxes was investigated.

• 1) By matching the ACh and tritium outflow on a molar basis, an average ratio of 100: 2–4 was found. When expressed as a percentage of tissue content, the ACh release at 2 Hz (2 min) was 4.1 in CN, 0.92 in CC and 0.44 in Th. Lower percent values in the same rank order, were found for tritium outflow with Phys. Thus, CN has the highest secretory activity.

• 2) Tritium evoked outflow in the presence of Phys was nearly halved in comparison with the normal values (without Phys). Therefore, the autoreceptor-mediated negative feed-back seems to be similar in the three areas.

• 3) Tritium evoked outflow in the presence of HC-3 was more than doubled in Th (less so in CC and CN) in comparison with the normal values. A second stimulation at 2 Hz (2 min) gave rise to the same outflow in Th while an evident fall in radiolabel efflux was found in CN. Therefore the blockade of high affinity choline uptake plays a different role in Th and CN.

• 4) The ratio between two subsequent periods of stimulation at 2 Hz for 2 min (at the 45^th and 75^th min, St₂/St₁) ranged as a rule between 0.75 and 1 for ACh and tritium. However, when the evoked outflow of ACh and tritium was tested during St₂ at different rates (1–2–5–10 Hz), a three-times greater increase of ACh with respect tritium was found, as a function of the stimulation frequency. This suggests an inverse relationship between specific radioactivity of released transmitter and second stimulus intensity, above all when Phys and HC-3 are not present.

• 5) GABA 0.3–0.6 mM, added before St₂, inhibited the evoked ACh and tritium efflux in CC and CN and increased the resting release in a dose-dependent manner. Conversely, picrotoxin, which prevented GABA effect, increased the evoked ACh release but not tritium efflux. Morphine 30 μM also inhibited the evoked ACh and tritium efflux from Th slices. However, the drug in the presence of naloxone enhanced ACh release but not tritium outflow.

• 6) In conclusion, the two methods give results only in part superimposable. Tritium method allows drug-induced inhibition to be seen more readly than facilitation. This fact may depend on the reduction of ACh specific radioactivity in St₂, whenever drug treatment enhances the release process, thus involving less labelled (or more diluted) neurotransmitter stores.