Projection-dependent VGluT1 and VGluT2 expression and terminal morphology in mouse visual circuits

Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) exhibit largely complementary distributions and have been proposed as molecular markers of descending modulatory and ascending driver-like pathways, respectively. However, this correspondence has rarely been tested directly in anatomically identified projections. We combined anterograde Phaseolus vulgaris leucoagglutinin tracing with simultaneous VGluT1 and VGluT2 immunofluorescence to characterize glutamatergic boutons arising from the primary and secondary visual cortices, lateral geniculate nucleus, lateral posterior thalamic nucleus, and superior colliculus in adult mice. Among 2,178 singly labelled boutons, VGluT1 predominated in corticothalamic, corticopontine, corticotectal, corticostriatal, and corticocortical feedback projections. Conversely, thalamocortical, tectothalamic, thalamostriatal, and tectopontine projections were almost exclusively VGluT2-positive. Projection direction did not fully predict transporter phenotype: ascending corticocortical projections remained predominantly VGluT1-positive, whereas the descending tectopontine projection was exclusively VGluT2-positive. Only 12 of 2,190 VGluT-immunoreactive boutons exhibited detectable VGluT1/VGluT2 colocalization. Morphometric analyses further revealed an interaction between VGluT isoform and projection direction. Within ascending projections, VGluT2-positive boutons and puncta were larger than their VGluT1-positive counterparts, whereas no isoform-related difference in bouton area was detected within descending projections. These findings demonstrate an association between VGluT phenotype and projection class in the mouse visual system. VGluT isoforms therefore provide informative markers of pathway organization, but neither transporter identity nor terminal size alone constitutes an invariant molecular indicator of projection direction or driver-modulator function.