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 rat inferior colliculus. Additional co-immunolabeling of microtubule-associated protein 2 and neuronal class III beta-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 axo-dendritic 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 VGLUT1 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 predominantly on the dendrites and VGLUT2 on both dendrites and somas.

Figure 1

Figure 1a–f: Low magnification view of immunolabeling for VGLUT1 (a,c,e) and VGLUT2 (b,d,f) immunolabeling in the central nucleus of the inferior colliculus (CIC) (a,b), layers 1,2 and 3 of the lateral cortex of the inferior colliculus (LCIC) (c,d,) and the dorsal cortex of the inferior colliculus (DCIC) (e,f). Bar = 20 microns.

Figure 2

Figure 2a–d: Co-immunolabeling for VGLUT1 (blue), VGLUT2 (green) and TUJ1/MAP2 (red) in the central nucleus of the inferior colliculus. Many VGLUT2 immunolabeled ending are seen surrounding the soma and proximal dendrites of medium to large and large neurons. Both VGLUT1 and VGLUT2 labeled puncta corresponding to axo-dendritic endings are seen throughout the neuropil. Bar = 20 microns.

Figure 3

Figure 3a–d: The photomicrograph from Figure 2a with immunolabeling for VGLUT1 (3a - blue), VGLUT2 (3b- green) and TUJ1/MAP2 (3c red) in the central nucleus of the inferior colliculus presented separately and then together as in Figure 2a. Asterisks mark two neurons receiving multiple VGLUT2 immunolabeled puncta on their soma. Bar = 20 microns.

Figure 4

Figure 4a–d: Co-immunolabeling for VGLUT1 (blue), VGLUT2 (green) and TUJ1/MAP2 (red) in the lateral cortex (a,b) and dorsal cortex (c,d) of the inferior colliculus. In the lateral cortex both VGLUT1 and VGLUT2 labeled puncta corresponding to axo-dendritic endings are seen throughout the neuropil. In the dorsal cortex many VGLUT2 immunolabeled ending are seen surrounding the somata and proximal dendrites of medium to large neurons. Both VGLUT1 and VGLUT2 immunolabeled puncta corresponding to axo-dendritic endings are seen throughout the neuropil. Bars = 20 microns