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. 2009 Oct 30;464(3):188-92.
doi: 10.1016/j.neulet.2009.08.042. Epub 2009 Aug 21.

Coincidence of neurokinin 1 receptor with the vesicular glutamate transporter 3 (VGLUT3) in the rat forebrain

K G Commons  1 , M R Serock
Affiliations

Coincidence of neurokinin 1 receptor with the vesicular glutamate transporter 3 (VGLUT3) in the rat forebrain

K G Commons et al. Neurosci Lett. .

Abstract

The third vesicular glutamate transporter (VGLUT3) is expressed in a subset of cholinergic and GABAergic neurons in the forebrain. In this study the distribution of VGLUT3 was mapped in relation to the receptor for substance P, neurokinin 1 (NK1), which has been independently reported within cholinergic and GABAergic neurons in a similar distribution. Dual immunofluorescence labeling techniques were used, sometimes in combination with triple labeling for the vesicular acetylcholine transporter (VAChT), to identify cholinergic cells. Virtually all cells immunolabeled for VGLUT3 in the nucleus accumbens core and shell regions, ventral pallidum, olfactory tubercle and caudate putamen were cholinergic and also contained immunolabeling for the NK1 receptor. In the hippocampal formation where VGLUT3 has been described in GABAergic neurons, colocalization between NK1 and VGLUT3 was also common but less complete. Cells double labeled for NK1 and VGLUT3 were most prevalent in stratum radiatum in the CA1 subfield. In the habenula VGLUT3 was also found within NK1 receptor immunolabeled neurons. However, there were some areas where neurons containing these two proteins were separate populations including the cerebral cortex and median raphe nucleus. These results reveal a trend for VGLUT3 to localize within neurons containing the NK1 receptor in several areas of the forebrain.

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Figures

Fig. 1
Fig. 1
Dual immunolabeling for VGLUT3 and NK1 in the basal forebrain nucleus accumbens core (AcbC) and shell (AcbSh) region show virtually all cells with immunolabeling VGLUT3 also contain immunolabeling for NK1 receptor, although additional cells with NK1 alone are present. A. VGLUT3 identifies scattered cells. A′. NK1 has a similar distribution. A″. Overlay of VGLUT3 (green) and NK1 (red). Boxed regions of core region shown at higher magnification in (B) and shell region shown in (C). B. VGLUT3 immunolabeling diffusely fills perisomatic region of cells, whereas NK1 immunolabeling defines the overlaying cytoplasm in dually immunolabeled cells (arrowheads). Additional cells that contain NK1 immunolabeling alone are detected (arrow). B′ and B″ show labeling for each antigen individually. C. In the AcbSh region NK1- and VGLUT3-immunolabeling are similarly colocalized. Bar in A″ = 250 um, B = 60 um, B and C same scale.
Fig. 2
Fig. 2
Cells dually labeled for NK1 and VGLUT3 are cholinergic in the basal forebrain and striatum. A. The same cells in each image (arrowheads) of the nucleus accumbens shell region have detectable VGLUT3 (A), NK1 (A′) and VAChT (A″), overlay of all three fluorophores with VGLUT3 (green), NK1 (red), and VAChT (fuchsia) in panel A‴. B. Similarly in the striatum, cholinergic cells with VGLUT3 also contain NK1 (arrowheads), while there are addition cells that contain NK1 alone (arrow in B′ and B‴. All panels same scale, bar in B‴ = 60 um.
Fig. 3
Fig. 3
NK1 and VGLUT3 were colocalized in the hippocampal formation (A-C) but not in the median raphe nucleus (D). A. A photomontage of three cells in the dentate gyrus that contain both NK1 outlining the cytoplasm (red) and VGLUT3 (green, arrow heads). The white line marks the junction between the granule cell layer (GCL) and the hilus, these cells are located just at the border in the subgranular layer. B. Two neighboring interneurons located in stratum radiatum of the CA1 region are dually labeled for NK1 and VGLUT3 (arrowheads). Both panels same scale bar = 20 um. C. Relative proportions of cells dually labeled for VGLUT3 and NK1 (filled circles) and cells containing only VGLUT3 (open circles) by laminar location and subfield, CA1, CA3 and dentate gyrus (DG). Several sections were sampled and single VGLUT3 labeled and cells dually labeled for VGLUT3 and NK1 were counted in each lamina. Medio-lateral distribution of cells is compressed and between subfield comparisons are not quantitative since each region was sampled independently from a different number of sections. For the CA1 region, most cells containing VGLUT3 were located in stratum radiatum (86% or 66/77) and the majority of these also contained NK1 immunolabeling (89% or 59/66). In all the remaining areas, the percentage of VGLUT3 cells that also contained NK1 averaged 44-62%, except for stratum radiatum of the CA3 region where 71% were dually labeled. Total number of cells sampled in CA1=96, CA3=19, and DG=120. Lamina abbreviations are O, oriens; P, pyramidale; L, lucidum; R, radiatum; M, molecular layer; sG, subgranular layer; H, hilus. D. Some areas exhibited very little colocalization between NK1 and VGLUT3 including the median raphe nucleus. VGLUT3 is present in large cells (arrowheads) that are distinct from NK1 immunolabeled cells in the same field (arrows) that are typically smaller in diameter. Scale bar = 40 um.
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