Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum

Abstract

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [ 3 H]choline and subsequent synthesis and release of [ 3 H]ACh were investigated in rat striatal segments. Incubation with [ 3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [ 3 H]choline and [ 3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [ 3 H]choline led predominantly to the accumulation of [ 3 H]ACh. Electrical stimulation and KCl depolarization selectively released [ 3 H]ACh but not [ 3 H]choline. [ 3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [ 3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [ 3 H] ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca 2+ dependent and exhibited slightly different sensitivities to N-and P-type Ca 2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [ 3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern

“…It has been reported that this increase is suppressed by 0.5 μM tetrodotoxin or by nominally Ca 2+ ‐free conditions, as reported previously (Muramatsu et al, 2017). HPLC analysis has demonstrated that the evoked efflux reflects the [ 3 H]ACh release from striatal cholinergic interneurons (Muramatsu et al, 2019, 2022). Figure 1a–d shows representative results in which stimulation with single pulses or multiple pulses at 0.5 Hz for 30 s (15 pulses) appeared to increase the [ 3 H]efflux.…”

“…In the present study, [ 3 H]ACh was preloaded in rat striatal cholinergic interneurons, and the release evoked by electrical stimulation was monitored under a condition in which presynaptic cholinergic and dopaminergic negative feedback was suppressed, and high‐ and low‐affinity choline transporters and postsynaptic ACh uptake were blocked (Muramatsu et al, 2022). Therefore, the [ 3 H]ACh released in this study was considered to be derived from [ 3 H]ACh that was synthesized during the preincubation with [ 3 H]choline before superfusion, and newly synthesized [ 3 H]ACh from cytosolic [ 3 H]choline during superfusion.…”

Non-targeted metabonomics and transcriptomics revealed the mechanism of mulberry branch extracts promoting the growth of Sanghuangporus vaninii mycelium

“…Androstenedione, a steroidal hormone, is thought to be an enhancer for athletic performance and build body muscles ( Badawy et al, 2021 ). Vesamicol, a selective vesicular acetylcholine transporter inhibitor, and acetylcholine can antagonistically regulate cholinergic transmission to treat cholinergic dysfunction-associated disorders ( Muramatsu et al, 2022 ). Results implied that the mycelium harvested from 0.02% MBE liquid culture might possess improved medicinal effects.…”

Presynaptic adenosine receptor heteromers as key modulators of glutamatergic and dopaminergic neurotransmission in the striatum

“…2), acetylcholine predominantly exerts a facilitation of dopamine release, although muscarinic acetylcholine autoreceptors should moderate acetylcholine release (Muramatsu et al, 2022). Through the α 4 -β 2 *-D 2 R/D 3 R heteromers and the D 2 R-D 4 R heteromers localized in the glutamatergic terminals and possibly D 2 R localized in cholinergic interneurons (Muramatsu et al, 2022) (Fig. 2), dopamine predominantly exerts an inhibition of dopamine release.…”

“…Through these heteromers localized in the dopaminergic terminals and, also, α 7 nAChR localized in the glutamatergic terminals (Kaiser and Wonnacott, 2000;Rassoulpour et al, 2005) (Fig. 2), acetylcholine predominantly exerts a facilitation of dopamine release, although muscarinic acetylcholine autoreceptors should moderate acetylcholine release (Muramatsu et al, 2022). Through the α 4 -β 2 *-D 2 R/D 3 R heteromers and the D 2 R-D 4 R heteromers localized in the glutamatergic terminals and possibly D 2 R localized in cholinergic interneurons (Muramatsu et al, 2022) (Fig.…”