Expression of the somatostatin subtype 2A receptor in the rabbit retina

Abstract

In the retina, somatostatin influences neuronal activity likely by acting at one or more somatostatin subtype (sst) receptors. Somatostatin and somatostatin-binding sites are distributed predominantly to the inner retina. The present study has investigated the cellular expression of one of the sst receptors, the sst2A receptor isoform, in the rabbit retina. These studies have used a new polyclonal antibody directed to the predicted C-terminus of mouse sst2A(361-369) receptor. Antibody specificity was tested by preadsorption of the primary antibody with a peptide corresponding to sst2A(361-369). sst2A Receptor immunoreactivity was localized mainly to the plasma membrane of rod bipolar cells and to sparsely occurring, wide-field amacrine cells. Immunostaining in rod bipolar cells was strongest in the axon and axon terminals in lamina 5 of the inner plexiform layer (IPL) and was weakest in the cell body and dendrites. Double-labeling experiments using a monoclonal antibody against protein kinase C (PKC; alpha and beta), a rod bipolar cell-selective marker, showed complete colocalization. In horizontal sections of retina, immunostained bipolar cell bodies had a dense distribution, which is in agreement with the reported distribution of rod bipolar cell bodies. Immunoreactive amacrine cell bodies were located at the border of the inner nuclear layer and the IPL, and thin varicose processes ramified mainly in laminae 2 and 4 of the IPL. These observations indicate that somatostatin influences visual information processing in the retina 1) by acting presynaptically on rod bipolar cell axon terminals and b) by influencing the activity of sparsely occurring amacrine cells.

Fig. 1

Localization of somatostatin subtype 2A (sst_2A) receptor immunoreactivity in the rabbit retina. A: The sst_2A receptor immunostaining was prominent in rod bipolar cells. B: Lack of sst_2A receptor immunostaining in a control section incubated with the sst_2A receptor antibody preadsorbed with 10⁻⁵ M sst_2A (361–369). OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 15 μm.

Fig. 2

Confocal image of sst_2A receptor-immunoreactive rod bipolar cell in a transverse section of retina. sst_2A Receptor immunoreactivity was closely associated with the plasma membrane of rod bipolar cells. The axon descended into the inner plexiform layer (IPL), where it terminated with a few large, club-like processes in lamina 5 of the IPL. sst_2A Receptor immunoreactivity was also associated with thin processes that ramified in laminae 2 and 4 of the IPL. These processes originated from sparsely occurring amacrine cells. OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. Z axis = 1 μm. Scale bar = 7.5 μm.

Fig. 3

The sst_2A receptor-immunostained cell bodies and axon terminals were found in the distal inner nuclear layer (INL) and proximal INL, respectively. Horizontal sections through the INL (A) showing sst_2A receptor-immunostained cell bodies and through the inner plexiform layer (IPL; B) showing sst_2A receptor-immunostained axon terminals. Scale bars = 25 μm in A, 15 μm in B.

Fig. 4

Coexpression of sst_2A receptor (A) and protein kinase C (PKC; B) immunoreactivities in rod bipolar cells. The sst_2A receptor showed complete coexpression with the rod bipolar, cell-selective marker PKC, further confirming that the sst_2A receptor is expressed by rod bipolar cells. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 15 μm.

Fig. 5

The sst_2A receptor-immunoreactive amacrine cells were sparsely occurring. Immunoreactive cell bodies were located at the border of the inner nuclear layer (INL) and the inner plexiform layer (IPL). They were likely to be multistratified and gave rise to the processes in laminae 2 and 4 of the IPL. In transverse sections, these processes in laminae 2 and 4 were not continuous. OPL, outer plexiform layer; GCL, ganglion cell layer. Scale bar = 25 μm.

Fig. 6

A–C: The sst_2A receptor-immunoreactive amacrine cells in a horizontal section of the retina. Strong immunostaining outlined these cell bodies and their main processes. Scale bar = 25 μm.

Fig. 7

Confocal image showing the localization of sst_2A receptor immunoreactivity closely associated with the plasma membrane of amacrine cells. Two different planes of focus through the same cell. Arrows indicate sst_za immunoreactivity associated with the plasma membrane in the cell body (A) and the cell body and processes (B). Z axis = 1 μm. Scale bar = 12.5 μm.

Fig. 8

Lack of colocalization of sst_2A receptor and tyrosine hydroxylase (TH) immunoreactivities. Composite image of sst_2A receptor (green) and TH (red) immunoreactivities in a transverse section of retina. Arrows indicate TH-immunoreactive processes. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 10 μm.

Fig. 9

Localization of sst_2A receptor (A) and TH (B) immunoreactivities in the same section of rabbit retina. A: The sst_2A receptor containing rod bipolar cells and amacrine cells. Note the amacrine cell body (arrow) and processes in laminae 2 and 4. B: TH-immunoreactive processes were localized to laminae 1, 3, and 5 of the IPL. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 25 μm.

Fig. 10

Schematic diagram illustrating the distribution of somatostatin and sst_2A receptor immunoreactivities in rabbit retina. Images of the rod bipolar cell and the somatostatin-immunoreactive amacrine cell were adapted from Rickman et al. (1996) and Gillette and Dacheux (1995). SRIF, somatotropin-release inhibiting factor; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.