Neurokinin 1 receptor expression in the rat retina

Abstract

Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the gamma-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine tells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina.

Fig. 1

Neurokinin 1 (NK1) immunofluorescence in retinal sections cut perpendicular to the vitreal surface. A: NK1-immunoreactive (IR) cells are mainly located in the proximal inner nuclear layer (INL), whereas their processes are densely distributed to all laminae of the inner plexiform layer (IPL). B: Rare NK1-IR cells (arrow) are observed in the outer INL. C,D: NK1-IR processes (arrows) running in the distal INL and arborizing in the outer plexiform layer (OPL). In C, the IPL laminae are numbered (1–5): the highest density of NK1-IR processes is in lamina 2. GCL, ganglion cell layer; ONL, outer nuclear layer. Scale bars = 50 µm in A and B, 30 µm in C and D.

Fig. 2

NK1 immunoreactivity in retinal sections cut parallel to the vitreal surface. Avidin-biotin-peroxidase technique. A: NK1-IR cell bodies in the INL. B,C: Photomicrographs taken from regions where the section was cut in an oblique plane, so that different retinal layers are present in the same section. Note NK1-IR processes (arrows) innervating and arborizing in the OPL. D: Section through the OPL showing the NK1-IR plexus. Scale bar = 30 µm.

Fig. 3

A: An NK1-IR cell (arrow) located in the GCL. The soma size of this cell is similar to that of NK1-IR cells in the INL, and it is likely to be a displaced amacrine cell. B: Higher magnification photomicrograph showing a putative NK1-IR displaced amacrine cell (small arrow) together with a larger NK1-IR cell in the GCL (large arrow), which is likely to be a ganglion cell. The open arrow points to a very faint NK1-IR cell in the distal INL. The location and morphology of this latter cell resemble those of a bipolar cell. Scale bars = 30 µm.

Fig. 4

Retinal sections cut parallel to the vitreal surface and processed by double-label immunohistochemistry by using antibodies directed against NK1 and γ-aminobutyric acid (GABA), and NK1 and tyrosine hydroxylase (TH). Left: NK1 immunoreactivity is shown in green; GABA immunoreactivity is shown in red; NK1+GABA shows the simultaneous visualization of NK1 (green) and GABA (red-orange) immunoreactivities in the same preparation with a dual-band filter. Note that most GABA-IR cell bodies also display NK1 immunoreactivity, which is confined to their plasma membrane. In addition, some NK1-IR cell bodies do not contain GABA immunoreactivity, and some GABA-IR cells do not express NK1 immunoreactivity. Right: TH (green) and NK1 (red) immunoreactivities visualized with fluorescein and rhodamine filters, respectively. Top two figures show two small TH-IR cell bodies displaying NK1 immunoreactivity at their cell surface; bottom two figures illustrate the colocalization of TH and NK1 immunoreactivities in a large cell (arrows point to the NK1 cell surface staining of such cell). Scale bars = 10 µm.

Fig. 5

A: Confocal microscopic images showing NK1 plasma membrane staining (green) of cells containing GABA immunoreactivity (red) and the colocalization of NK1 + GABA immunoreactivities in the same preparation. Arrows point to examples of cells expressing both NK1 and GABA. B: Confocal microscopic images showing NK1 cell surface staining (red) of a cell containing TH immunoreactivity (green) and the colocalization of NK1+TH immunoreactivities in the same preparation. Arrows point to the NK1 cell surface staining. Scale bars = 10 µm.

Fig. 6

Schematic reconstruction made from photographic negatives (T-MAX 400) of NK1- and GABA-IR profiles, respectively. C was obtained by superimposing (A) and (B); filled profiles represent cells displaying both NK1 and GABA immunoreactivities, stippled profiles represent cells only having NK1 immunoreactivity, and open profiles represent cells displaying only GABA immunoreactivity.

Fig. 7

A: TK immunoreactivity in a retinal section. TK-IR cells are located in the proximal INL. Their processes arborize mainly in laminae 1 and 5 (arrowheads) of the IPL and some fine immunostained processes are also in lamina 3. B: NK1-IR processes are densely distributed throughout the IPL. Scale bar = 30 µm.

Fig. 8

A: MAb 115A10-IR cell bodies are mainly confined to the distal INL, and their processes are densely distributed to both the OPL and IPL. B: NK1-IR somata are located in more proximal regions of the INL, and their position does not overlap with that of MAb 115A10-IR somata. Scale bar = 20 µm.