Differential localization of vesicular glutamate transporters and peptides in corneal afferents to trigeminal nucleus caudalis

Abstract

Trigeminal afferents convey nociceptive information from the corneal surface of the eye to the trigeminal subnucleus caudalis (Vc). Trigeminal afferents, like other nociceptors, are thought to use glutamate and neuropeptides as neurotransmitters. The current studies examined whether corneal afferents contain both neuropeptides and vesicular glutamate transporters. Corneal afferents to the Vc were identified by using cholera toxin B (CTb). Corneal afferents project in two clusters to the rostral and caudal borders of the Vc, regions that contain functionally distinct nociceptive neurons. Thus, corneal afferents projecting to these two regions were examined separately. Dual immunocytochemical studies combined CTb with either calcitonin gene-related peptide (CGRP), substance P (SP), vesicular glutamate transporter 1 (VGluT1), or VGluT2. Corneal afferents were more likely to contain CGRP than SP, and corneal afferents projecting to the rostral region were more likely to contain CGRP than afferents projecting caudally. Overall, corneal afferents were equally likely to contain VGluT1 or VGluT2. Together, 61% of corneal afferents contained either VGluT1 or VGluT2, suggesting that some afferents lack a VGluT. Caudal corneal afferents were more likely to contain VGluT2 than VGluT1, whereas rostral corneal afferents were more likely to contain VGluT1 than VGluT2. Triple-labeling studies combining CTb, CGRP, and VGluT2 showed that very few corneal afferents contain both CGRP and VGluT2, caudally (1%) and rostrally (2%). These results suggest that most corneal afferents contain a peptide or a VGluT, but rarely both. Our results are consistent with a growing literature suggesting that glutamatergic and peptidergic sensory afferents may be distinct populations.

Figure 1

Light micrographs show the distribution of peroxidase-labeled CTb-containing corneal afferents (arrows) at the caudal (A) and rostral (B) boundaries of ventrolateral Vc (diagram insets). The black box on each diagram delineates the ventrolateral regions of Vc/C1 (A) and Vi/Vc (B) that were imaged for analysis. Representative diagrams of the caudal and rostral Vc regions are modified from the digital atlas of Paxinos and Watson (Paxinos and Watson, 1998) and reproduced here with permission from the publisher. The tissue was incubated in goat anti-CTb primary antibody (1:25,000; List Biological Laboratories, Inc.) as described in the Methods section. Bound primary antibody was visualized by incubating tissue sections in biotinylated horse anti-goat secondary antibody (1:400; Vector Laboratories, Inc., Burlingame, CA) followed by incubation in Avidin-Biotin (Elite Vectastain ABC kit; Vector) and then in diaminobenzidine-hydrogen peroxide solution. Tissue was mounted on gelatin-coated slides, dehydrated and then coverslipped with DPX mounting medium (Sigma). Images were taken with on an Olympus BX51 microscope equipped with a DP71 camera (Olympus America, Inc, Center Valley, PA) and were adjusted for optimal brightness and contrast using Adobe Photoshop (Adobe Systems Inc., San Jose, CA). Sp5C = spinal trigeminal subnucleus caudalis, Sp5I = spinal trigeminal subnucleus interpolaris, spV = spinal trigeminal tract. Scale bars = 200 μm.

Figure 2

Confocal micrographs show the distribution of corneal afferents labeled with CTb (A, D; red) and nociceptive CGRP-containing afferents (B, E; green) at the caudal (A-C) and rostral (D-F) boundaries of ventrolateral Vc. Some CTb-labeled varicosities also contained CGRP (arrows). spV = spinal trigeminal tract. Scale bars = 20 μm.

Figure 3

High magnification confocal micrographs show the distribution of corneal afferents labeled with CTb (A; red) and CGRP (B; green) in Vi/Vc. An overlay image (C) shows that a subset of CTb-labeled varicosities also contain CGRP-ir (arrows). Co-localized profiles appear yellow in the overlay. bars = 5 μm.

Figure 4

The summary graph shows the percentages of CTb-labeled corneal afferents that also contained either CGRP or SP immunoreactivity across four animals. Total percentages represent the sum of CGRP- or SP-labeled corneal afferents found caudally in Vc/C1 and rostrally in Vi/Vc. Statistics were used to compare the proportions of CGRP-containing varicosities to total CTb-labeled varicosities in Vc/C1 and Vi/Vc. There was a significant difference in this proportion between the two regions (z = 2.663, P = 0.008). There was no such difference when the proportion of SP-containing to total CTb-labeled varicosities in Vc/C1 and Vi/Vc were compared (z = 0.359, P = 0.720).

Figure 5

Confocal micrographs of corneal afferents labeled with CTb-ir (red) and VGluT1-ir (green) at the ventrolateral Vc/C1 (A) and Vi/Vc (B) transition regions. CTb-labeled corneal afferent fibers (red) are found in lamina I and II in Vc/C1 and are more dispersed at the rostral Vi/Vc boundary. VGluT1 labeling (green) is found in lamina I and II inner (IIi) in Vc/C1 (A) and diffusely throughout the ventrolateral Vi/Vc (B). CTb-labeled varicosities containing VGluT1-ir are more abundant in Vi/Vc (B). Co-localized profiles appear yellow and a few are indicated by arrows. spV = spinal trigeminal tract. Scale bars = 50 μm.

Figure 6

The summary graph shows the percentages of CTb-labeled corneal afferents that also contained one of the VGluTs across five animals. The percentages in the Total columns represent the sum of VGluT1- or VGluT2-labeled corneal afferents in both caudal Vc/C1 and rostral Vi/Vc. We compared the proportions of VGluT1-containing to total CTb-labeled varicosities in Vc/C1 and Vi/Vc and found that there was a significant difference in this proportion between these two regions (z = 8.050, P < 0.001). There was also a significant difference in the proportions of VGluT2-containing to total CTb-labeled varicosites in Vc/C1 and Vi/Vc (z = 2.074, P = 0.038).

Figure 7

Confocal micrographs of CTb (A, E, white), CGRP (B, F, red) and VGluT2 (C, G, green) at the caudal (Vc/C1) (A - D) and rostral (Vi/Vc) (E - H) boundaries of trigeminal subnucleus caudalis. All three markers were present in the superficial laminae of ventrolateral Vc/C1 (D) and were more dispersed in the ventrolateral region of Vi/Vc (H). spV = spinal trigeminal tract. Scale bars = 20μm.