Origins of Glutamatergic Terminals in the Inferior Colliculus Identified by Retrograde Transport and Expression of VGLUT1 and VGLUT2 Genes

Abstract

Terminals containing vesicular glutamate transporter (VGLUT) 2 make dense axosomatic synapses on tectothalamic GABAergic neurons. These are one of the three types of glutamatergic synapses in the inferior colliculus (IC) identified by one of three combinations of transporter protein: VGLUT1 only, VGLUT2 only, or both VGLUT1 and 2. To identify the source(s) of these three classes of glutamatergic terminals, we employed the injection of Fluorogold (FG) into the IC and retrograde transport in combination with in situ hybridization for VGLUT1 and VGLUT2 mRNA. The distribution of FG-positive soma was consistent with previous reports. In the auditory cortex, all FG-positive cells expressed only VGLUT1. In the IC, the majority of FG-positive cells expressed only VGLUT2. In the intermediate nucleus of the lateral lemniscus, most FG-positive cells expressed VGLUT2, and a few FG-positive cells expressed both VGLUT1 and 2. In the superior olivary complex (SOC), the majority of FG-positive cells expressing VGLUT2 were in the lateral superior olive, medial superior olive, and some periolivary nuclei. Fewer FG-positive cells expressed VGLUT1&2. In the ventral cochlear nucleus, almost all FG-positive cells expressed VGLUT1&2. On the other hand in the dorsal cochlear nucleus, the vast majority of FG-positive cells expressed only VGLUT2. Our data suggest that (1) the most likely sources of VGLUT2 terminals in the IC are the intermediate nucleus of the lateral lemniscus, the dorsal cochlear nucleus, the medial and lateral superior olive, and the IC itself, (2) VGLUT1 terminals in the IC originate only in the ipsilateral auditory cortex, and (3) VGLUT1&2 terminals in IC originate mainly from the VCN with minor contributions from the SOC and the lateral lemniscal nuclei.

Keywords: auditory system; in situ hybridization; retrograde transport; vesicular glutamate transporter.

Figure 1

Injection sites of FG. Top images show the center of the injection site in three animals processed for fluorescent ISH. Line drawings show the full extent of the injection sites (gray) at several rostro-caudal levels. In case 08-110 (A), the injection is almost restricted to the central nucleus (ICC). In cases 08-118 (B) and 08-124 (C), the injections are larger, and they cover both the lateral cortex (LC) and lateral part of the ICC. Numbers indicate the distance (in millimeters) from the section which contains the most rostral part of the ventral nucleus of the lateral lemniscus (VNLL). Note that in the case 08-110, two injections were made. Scale bar: 1 mm.

Figure 2

Plots of retrogradely labeled cells in the IC (A), NLL, SOC, and CNC (B) of the case 08-118. In the IC (A), contralateral side to the injection site was presented. Red dots indicate cells positive for both FG and VGLUT2 but negative for VGLUT1 (FG+/ V1−/ V2+). Green dots indicate cells positive for all three molecules (FG⩲/ V1⩲/ V2⩲). Blue dots indicate cells positive for FG but negative for VGLUT1 and VGLUT2 (FG+/ V1−/ V2−). Numbers indicate the distance (in millimeter) from the section which contains the most rostral part of the VNLL. Scale bar: 2 mm.

Figure 3

Fluorescent micrographs of retrogradely labeled cells in the IC (A), NLL (B–D), CNC (E–G), and SOC (H–Q). Some retrogradely labeled cells expressed only VGLUT2 (white arrows) or both VGLUT1 and VGLUT2 (black arrows). The “c” and “i” after the abbreviation of the nuclei mean contralateral and ipsilateral to the injection site. Green: VGLUT2; red: VGLUT1; blue: FG. Scale bar: 50 μm.

Figure 4

Corticofugal neurons in layer 5 (V) express only VGLUT1. Brightfield ISH for VGLUT1 (A,C) or VGLUT2 (B,D) combined with immunohistochemistry for FG showed that retrogradely labeled pyramidal neurons express VGLUT1 but not VGLUT2. (C,D) Are the higher magnification of boxes in (A,B), respectively. Scale bars: 500 μm (A,B), and 50 μm (C,D).

Figure 5

Weak expression of VGLUT2 in layer 6 (VI) of rostral auditory cortex in same case as Figure 4. Compared to Figure 4B, this section is developed to show weak expression and consequently some background labeling. Modest VGLUT2 expression is in layer 6 of ipsilateral cortex (A) and contralateral cortex (B), but it is weak compared to the thalamus of the same section (C). Labeling in layers 2/3 and 4 (II/III and IV) is slightly above background and less intense than layer 6. Lack of above background label in layer 5 suggests no FG + neurons are VGLUT2 positive. Scale bar: 500 μm.