Different isoforms of a vesicular glutamate transporter (VGLUT) mediate 
glutamate uptake into synaptic vesicles of excitatory neurons. There is 
agreement that the VGLUTs are differentially expressed in brain, and that two 
isoforms, VGLUT1 and VGLUT2, are localized to excitatory axon terminals in the 
cerebellar cortex. While granule cells express solely VGLUT1, there is no report 
about the VGLUT(s) of the unipolar brush cell (UBC), the second type of 
glutamatergic interneuron residing in the cerebellar granular layer. In the 
mouse, UBCs are particularly numerous in the uvula (lobule IX) and nodulus 
(lobule X). These folia contain two distinct subsets of UBCs: one kind expresses 
the calcium-binding protein calretinin (CR), and the other kind expresses the 
metabotropic glutamate receptor (mGluR) 1alpha. UBCs give rise to an extensive 
system of intrinsic mossy fibers (MF), whose terminals innervate granule cells 
and other UBCs, altogether similar to those formed by the extrinsic MFs. The 
presence of both extrinsic and intrinsic MFs in the vestibulocerebellum makes it 
difficult to determine which type of VGLUT is contained in MFs formed by the UBC 
axons. Hence, the nodulus was isolated from sagittal cerebellar slices from 
postnatal day 10 mice, and cultured for 15-20 days in vitro. Double 
immunofluorescence and confocal microscopy showed that mossy terminals of 
CR-positive (CR(+)) UBCs were immunoreactive for VGLUT1 and VGLUT2, while mossy 
terminals of mGluR1alpha-positive (mGluR1alpha(+)) UBCs were provided with 
VGLUT1 only. Moreover, CR(+) dendritic brushes were contacted by mossy terminals 
provided with both transporters, while mGluR1alpha(+) dendritic brushes were 
contacted by mossy terminals immunopositive for VGLUT1 and immunonegative for 
VGLUT2. These data indicate that the two UBC subsets use different modalities of 
vesicular glutamate storage and form separate networks. We consider it possible 
that expressions of CR with VGLUT1/VGLUT2 and mGluR1alpha(+) with VGLUT1 in the 
two subsets of vestibulocerebellar UBCs are determined by specific vestibular 
inputs, carried by groups of primary and/or secondary vestibular afferents.