Immunolocalization of vesicular glutamate transporters 1 and 2 in the rat inferior colliculus

Abstract

The inferior colliculus is a major relay nucleus in the ascending auditory pathways that receives multiple glutamatergic inputs. Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) most often have complementary non-overlapping distributions and can be used to differentiate glutamatergic inputs. The present study therefore examined co-immunolabeling of VGLUT1 and VGLUT2 in three divisions of the inferior colliculus. Additional co-immunolabeling of microtubule associated protein 2 and neuronal class III β-tubulin provided visualization of neuronal soma and processes and allowed identification of axo-somatic versus axo-dendritic contacts. Results showed numerous VGLUT1 and 2 immunolabeled terminals in the central nucleus, lateral cortex and dorsal cortex. In all three divisions there was little to no co-containment of the two vesicular glutamate transporters indicating a complementary distribution. VGLUT1 made predominantly axo-dendritic connections in the neuropil, while VGLUT2 had many axo-somatic contacts in addition to axodendritic contacts. VGLUT2 immunolabeled terminals were numerous on the soma and proximal dendrites of many medium-to-large and large neurons in the central nucleus and medium to large neurons in the dorsal cortex. There were more VGLUT2 terminals than VGLUT 1 in all divisions and more VGLUT2 terminals in dorsal and lateral cortices than in the central nucleus. This study shows that VGLUT1 and VGLUT2 differentiate complementary patterns of glutamatergic inputs into the central nucleus, lateral and dorsal cortex of the inferior colliculus with VGLUT1 endings VGLUT1 endings predominantly on the dendrites and VGLUT2 on both dendrites and somas. Keywords auditory; auditory brain stem; co-localization Glutamate is the major excitatory transmitter in the ascending auditory pathway, used by multiple neurons and multiple projections at all the different levels. The presence of vesicular glutamate transporters (VGLUTs) which mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles has proven to be a useful immunocytochemical marker for glutamatergic terminals. There are at least three subtypes of VGLUTs: VGLUT1, VGLUT2 and VGLUT3. VGLUT1 and VGLUT2 are both associated with glutamatergic terminals and their localizations are generally complementary without overlap (e.g. Hioki et al, 2003; The inferior colliculus (IC) receives multiple inputs from ascending and descending auditory pathways as well as from non-auditory inputs (Adams 1979, 1980, 1983, Aitkin et al, 1981Bajo and Moore, 2005;Cant & Benson, 2006;Jain & Shore, 2006;Loftus et al, 2004;Malmierca et al, 2005;Marsh et al, 2002;Merchan et al, 1994;Moore et al, 1998; Okayama et al, 2006;Oliver, 2000;Oliver et al, 1997; Osen, 1972;Saldana et al, 1996; Shofield, 2002; Shofield and Cant, 1996; Zhou, 2006, Tokunaga et al, 1984;Zhou and Shore, 2006) many of which are glutamatergic (Malmierca, 2003 for review;Alibardi, 1998;Feliciano and Potashner, 1995;Ross et al, 1995;Saint Marie, 1996, Suneja et al, 1995....

Vesicular glutamate transporters: spatio-temporal plasticity following hearing loss

“…The discrepancy of vGluT1 and -2 co-localization in our study and the complementary distribution of vGluT1 and -2 in the CN granule cell region observed by others may be due to species differences (guinea pig in previous studies and rat in the current study). Recently, several studies of the auditory system suggest co-production of vGluT1 and -2 in the SOC (Billups, 2005; Blaesse et al, 2005) and the IC (Altschuler et al, 2008) under normal hearing conditions.…”

“…Although roles for vGluT1 and -2 in auditory brainstem plasticity have been suggested (Zhou et al, 2007; Altschuler et al, 2008; Zeng et al, 2009), vGluT3 function in this pathway has not been established. In addition to being profoundly deaf, mice with no vGluT3 expression exhibit decreased cochlear nucleus volumes and hyperexcitable neurons (Seal et al, 2008), similar to what is observed in the auditory pathways following hearing loss (Niparko and Finger, 1997; Kaltenbach and Afman, 2000; Kaltenbach et al, 2000; Mossop et al, 2000; Salvi et al, 2000; Brozoski et al, 2007; Bauer et al, 2008).…”

The auditory corticocollicular system: Molecular and circuit-level considerations

“…Given that CC cells likely employ VGLUT1(Ito and Oliver 2010), it is conceivable that the lack of VGLUT1 labeling is due to a paucity CC terminals in this region. In contrast, circumferential axo-somatic VGLUT2 labeling, thought to be only on giant GABAergic tectothalamic neurons (Ito, Bishop and Oliver 2009), was seen in layer 2, not layers 1 and 3 (Altschuler et al 2008) and appears to be seen preferentially in the extramodular parts of layer 2.…”

“…Distinctly higher expression of the vesicular inhibitory amino acid transporter is found in layer 2 of the LC, a result which is consistent with studies showing GAD staining in layer 2 of the LC (Ito, Bishop and Oliver 2011). In addition, for the vesicular glutamate transporters, the labeling of VGLUT1 is densest in the DC and although less dense in the CNIC and LC (Altschuler et al 2008), the lack of labeling in layer 2 of the LC is remarkable. Given that CC cells likely employ VGLUT1(Ito and Oliver 2010), it is conceivable that the lack of VGLUT1 labeling is due to a paucity CC terminals in this region.…”

Computer-assisted 3-D reconstructions of Golgi-impregnated neurons in the cortical regions of the inferior colliculus of rat

“…For example, recent studies have shown a majority of large neurons in the IC that are GABAergic (Oliver et al, 1994;Merchan et al, 2005;Hernández et al, 2006). Furthermore, the largest of these GABAergic ones also exhibit VGLUT2 axosomatic inputs (Altschuler et al, 2008;Ito et al, 2009). This is true not only for the CNIC neurons but also for the LCIC and DCIC (Ito et al, 2009) suggesting that they might correspond to the larger neurons (bitufted and chandelier types) described in the LCIC or the large multipolar neurons from the deep DCIC.…”