In vitro characterization of [3H]VAT in cells, animal and human brain tissues for vesicular acetylcholine transporter

Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [F-18]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [H-3]VAT, a tritiated counterpart of [F-18]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [H-3]VAT with Kd = 6.5 nM and B-max = 22.89 pmol/mg protein. In the [H-3]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [H-3]VAT was observed using VAChT ligands, the Ki values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [H-3]VAT autoradiography of rat brain sections showed strong signals in the striatum, moderate to high signals in vermis, thalamus, cortex, and hippocampus, and weak signals in cerebellum. Strong [3H]VAT ARG signals were also observed from striatal sections of normal nonhuman primates and human brains. Competitive ARG study with human striatal sections demonstrated strong ARG signals of [3H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (-)-vesamicol, but not by the sigma(1) receptor ligand Yun-122. ARG study also indicated that signal in the striatal sections from PSP human brains was lower than normal human brains. These data provide solid evidence supporting [F-18]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo.

Automated summary

The vesicular acetylcholine transporter (VAChT) plays a crucial role in the cholinergic system and alterations in it are associated with neurodegenerative disorders. A PET imaging tracer, [F-18]VAT, was recently developed to target VAChT in vivo with high affinity and selectivity. In vitro characterization of the tritiated counterpart of [F-18]VAT, [H-3]VAT, showed a saturated binding curve with VAChT-rich cell membrane extracts and binding inhibition by VAChT ligands, supporting [F-18]VAT as a suitable PET radiotracer for quantitatively assessing VAChT levels in vivo.