VGLUT2 mRNA and protein expression in the visual thalamus and midbrain of prosimian galagos (Otolemur garnetti)

Abstract

Vesicular glutamate transporters (VGLUTs) control the storage and presynaptic release of glutamate in the central nervous system, and are involved in the majority of glutamatergic transmission in the brain. Two VGLUT isoforms, VGLUT1 and VGLUT2, are known to characterize complementary distributions of glutamatergic neurons in the rodent brain, which suggests that they are each responsible for unique circuits of excitatory transmission. In rodents, VGLUT2 is primarily utilized in thalamocortical circuits, and is strongly expressed in the primary sensory nuclei, including all areas of the visual thalamus. The distribution of VGLUT2 in the visual thalamus and midbrain has yet to be characterized in primate species. Thus, the present study describes the expression of VGLUT2 mRNA and protein across the visual thalamus and superior colliculus of prosimian galagos to provide a better understanding of glutamatergic transmission in the primate brain. VGLUT2 is strongly expressed in all six layers of the dorsal lateral geniculate nucleus, and much less so in the intralaminar zones, which correspond to retinal and superior collicular inputs, respectively. The parvocellular and magnocellular layers expressed VGLUT2 mRNA more densely than the koniocellular layers. A patchy distribution of VGLUT2 positive terminals in the pulvinar complex possibly reflects inputs from the superior colliculus. The upper superficial granular layers of the superior colliculus, with inputs from the retina, most densely expressed VGLUT2 protein, while the lower superficial granular layers, with projections to the pulvinar, most densely expressed VGLUT2 mRNA. The results are consistent with the conclusion that retinal and superior colliculus projections to the thalamus depend highly on the VGLUT2 transporter, as do cortical projections from the magnocellular and parvocellular layers of the lateral geniculate nucleus and neurons of the pulvinar complex.

Figure 1

Coronal brain sections through the caudal thalamus of a galago. Sense and anti-sense probes for VGLUT2 confirm staining specificity for VGLUT2 mRNA and lack of secondary reactivity due to staining techniques. A) Anti-sense VGLUT2 probe stains VGLUT2 mRNA in the thalamus. B) Sense VGLUT2 probe does not stain VGLUT2 mRNA and does not show any secondary signal in the thalamus. Scale bar is 1 mm. The thalamic midline is to the right.

Figure 2

Serial sections through part of the rostral lateral geniculate nucleus (LGN) stained for (A) cytochrome oxidase (CO), (B) Nissl, (C) VGLUT2 protein and (D) VGLUT2 mRNA. Scale bar is 0.5 mm. Coronal sections, lateral is right.

Figure 3

Serial sections through the caudal lateral geniculate nucleus (LGN) stained for (A) cytochrome oxidase (CO), (B) Nissl, (C) VGLUT2 protein and (D) VGLUT2 mRNA. Scale bar is 0.5 mm. Coronal sections, lateral is left.

Figure 4

VGLUT2 mRNA (A–E) and protein (F–J) expression in each layer of the LGN. Scale bar is 100 µm.

Figure 5

LGN cell types can be differentiated according to pattern of staining for VGLUT2 mRNA. A) M cells are large and exhibit strong nuclear staining for VGLUT2 mRNA. B) P cells are slightly smaller but also show intense staining for VGLUT2. C) K cells are the smallest of all three and show weak, diffuse staining for VGLUT2. Scale bar is 50 µm.

Figure 6

Laminar pattern of VGLUT2 immunoreactivity across the LGN. A) VGLUT2 is strongly expressed in each layer of the LGN but less so in the interlaminar zones. However, high magnification (B) shows VGLUT2-positive terminals throughout the interlaminar zones. C) VGLUT2-positive terminals encircle labeled and unlabeled cells in the M layers of the LGN, but not in the P or K layers. Scale bar is (A) 250 µm and (B–C) 100 µm.

Figure 7

Serial sections through the pulvinar complex stained for (A) Nissl, (B) CO, (C) VGLUT2 mRNA and (D) VGLUT2 protein. Scale bar is 1 mm. Coronal sections; medial is left. PM, PL, PI: medial, lateral, and inferior divisions of the pulvinar complex.

Figure 8

High magnification images of VGLUT2 mRNA expression in each division of the pulvinar complex. A) PM and PL both show intense staining for VGLUT2 mRNA but (B) PL shows a denser distribution of VGLUT2-positive cells than PM. C) PI stains variably in density and intensity for VGLUT2 mRNA, indicated multiple populations of glutamatergic cells in this region. Scale bar is 250 µm.

Figure 9

VGLUT2 protein expression in the pulvinar is largely confined to cell bodies and processes instead of terminals. Scale bar is 25 µm.

Figure 10

Patchy distribution of VGLUT2 positive terminals in the pulvinar complex. Serial sections of the pulvinar complex in low magnification (A–C) and higher magnification (D–F) stained for CO (A, D), Nissl (B, E), and VGLUT2 protein (C, F). VGLUT2 staining shows a region between PM and PL with patches of glutamatergic terminals. These could be projections from the ISGS of the SC to the pulvinar. Scale bar is 1 mm (A–C) and 0.5 mm (D–F).

Figure 11

Diffential expression of VGLUT2 protein in each subdivision of the pulvinar complex. A) PM shows dense staining of VGLUT2-positive cell bodies. B) The medial region of PL shows dense patches of VGLUT2 positive terminals, which could be SC projections to pulvinar. C) Lateral PL shows dense VGLUT2 staining of cell bodies and sparse staining of terminals. D) Most of PI shows diffuse staining of VGLUT2 cell bodies but lacks VGLUT2 positive terminals. Scale bar is 100 µm.

Figure 12

Serial sections through the superior colliculus (SC) stained for (A) CO, (B) Nisl, (C) VGLUT2 mRNA and (D) VGLUT2 protein. Scale bar is 0.5 mm. Coronal sections; medial is right.