Cholecystokinin-like immunoreactive amacrine cells in the rat retina

Abstract

High levels of endogenous cholecystokinin (CCK) are present in the rat retina (Eskay & Beinfeld, 1982), but the cellular localization and physiological actions of CCK in the rat retina are uncertain. The goals of this study were to characterize the cells containing CCK, identify cell types that interact with CCK cells, and investigate the effects of CCK on rod bipolar cells. Rat retinas were labeled with antibody to gastrin-CCK (gCCK) using standard immunofluorescence techniques. Patch-clamp methods were used to record from dissociated rod bipolar cells from rats and mice. Gastrin-CCK immunoreactive (-IR) axons were evenly distributed throughout the retina in stratum 5 of the inner plexiform layer of the rat retina. However, the gCCK-IR somata were only detected in the ganglion cell layer in the peripheral retina. The gCCK-IR cells contained glutamate decarboxylase, and some of them also contained immunoreactive substance P. Labeled axons contacted PKC-IR rod bipolar cells, and recoverin-IR ON-cone bipolar cells. CCK-octapeptide inhibits GABA(C) but not GABA(A) mediated currents in dissociated rod bipolar cells.

Fig. 1. Gastrin CCK-IR amacrine cells in the rat retina

A: Varicose gCCK-IR axons are seen in a vertical section of the inner 20–25% of the inner plexiform layer (IPL). There is punctate labeling in a cell body (arrow) in the ganglion cell layer (GCL). Larger varicosities (arrowheads), probably on dendrites, are also seen in the middle of the IPL. Stack = 8 × 0.5 µm. (INL, inner nuclear layer). B: A meshwork of varicose gCCK-IR processes is seen in the far periphery of a whole mount. A single gCCK-IR cell body (arrow) is seen. Stack = 25 × 0.5 µm. C: The gCCK-IR axons are seen at higher magnification in central retina from a whole mount preparation. Stack = 8 × 0.5 µm. Scale bars = 10 µm (A) and 50 µm (B & C).

Fig. 2. Gastrin CCK-IR amacrine cells do not contain tyrosine hydroxylase (TH) or glycine transporter-1 (Glyt-1), and the gCCK-IR varicosities costratify with parvalbumin immunoreactivity

A: The TH-IR (green) processes are dense in the outer stratum of the IPL; there is also a sparse plexus of TH-IR processes in the center of the IPL. The large TH-IR amacrine cell soma is seen in the typical position in the inner row of somata in the INL. A gCCK-IR (red) amacrine cell soma (arrow) is shown in the GCL in peripheral retina. Neither the varicose gCCK-IR processes nor the perikarya contain TH-IR. Stack = 13 × 0.5 µm. B,C: Glyt-1 immunoreactivity (green) is present in processes throughout the IPL, as well as in a population of amacrine cells in the INL. The gCCK-IR (red) processes are associated with some of the Glyt-1 processes in the inner stratum of the IPL. Neither the perikaryon (B, arrow) nor the gCCK-IR processes contain Glyt-1 immunoreactivity. Glyt-1-IR perikaryon (C, arrowhead) in the ganglion cell layer did not contain gCCK immunoreactivity. Stack = 3 × 0.5 µm (B) and 7 × 0.5 µm (C). D: The parvalbumin-IR (green) perikarya in the inner nuclear layer (INL) are mostly AII-like amacrine cells, with dendrites in the inner and outer strata of the inner plexiform layer (IPL). There are also fainter parvalbumin-IR somata in the ganglion cell layer (GCL). Many of the gCCK-IR (red) varicosities are found in the vicinity of parvalbumin-IR processes in the inner stratum of the IPL. Stack = 5 × 0.5 µm. Scale bars = 10 µm (A, B, & C) and 20 µm (D).

Fig. 3

Gastrin CCK-IR amacrine cells are GABAergic. In a single optical section (0.5 µm), glutamate decarboxylase 65 (GAD) immunoreactivity (green) is seen throughout the IPL, with the highest levels in stratum 1, a narrow band at stratum 203 and a wider, more densely labeled band in stratum 5. There are also faintly GAD-IR somata in both the INL and GCL. The gCCK-IR (red) soma (arrow) clearly contains GAD immunoreactivity (green); the channels are combined on the right. The majority of the gCCK-IR processes appear to also contain GAD immunoreactivity. Scale bar = 10 µm.

Fig. 4. Gastrin CCK-IR processes contact rod bipolar axon terminals

A: Gastrin CCK-IR (red) varicose processes are found in stratum 5 in the vicinity of the protein kinase C-IR (green, PKC-IR) axon terminals in vertical section. Stack = 10 × 0.5 µm. B: At a higher magnification of a vertical section, the gCCK-IR varicosities contact the PKC-IR axon terminals (arrows). Single optical section = 0.5 µm. C: The contacts (arrows) between the gCCK-IR varicosities and the PKC-IR rod bipolar cell terminals are seen more clearly in a single optical section (0.5 µm) from whole-mount preparation. Scale bars = 25 µm (A), 5 µm (B), and 10 µm (C).

Fig. 5. CCK effect on GABA-induced currents in rod bipolar cells from mouse retina

A: Currents induced by 100-ms puff application of GABA in absence (black traces) or presence (grey traces) of 100 nM CCK with a sample rate of 10 ms. For these experiments, GABA(30 µM) was included in the puff-pipette alone (left pair of current traces), or together with TPMPA (100 µM, middle pair of current traces) or Bicuculine (100 µM, right pair of current traces). This data was obtained from three different cells. B: Histogram representation of the effect of CCK on the GABA_C-mediated current. Current amplitudes were normalized and the number of recorded cells is indicated above each bar representation. *** indicates statistical significance, P < 0.001.

Fig. 6. Gastrin CCK-IR processes contact ON-cone bipolar cell axon terminals

A: Type 2 and 8 cone bipolar cells are labeled with antisera to recoverin (green) in a vertical section. The ON-cone bipolar cells (type 8) have axon terminals in the vicinity of the gCCK-IR (red) varicosities in stratum 5. Stack = 7 × 0.5 µm. B: Contacts between the gCCK-IR varicosities and recoverin-IR ON-cone bipolar cells are seen at a higher magnification of a vertical section (arrows). Single optical section = 0.5 µm. C: The contacts (arrows) between gCCK-IR varicosities and recoverin-IR ON-cone bipolar cell terminal are seen more clearly in a single optical section (0.5 µm) in stratum 5 of the IPL. Scale bars=25 µm (A) and 10 µm (B & C).

Fig. 7. Some gCCK-IR (red) processes contain substance P-like immunoreactivity (green)

A: Substance P-IR processes in a vertical section are in narrow bands in strata 1 and 3, as well as a much denser band throughout stratum 5 of the inner plexiform layer (IPL). There are cell bodies in both the inner nuclear (INL) and ganglion cell layer (GCL). Some of the gCCK-IR processes in stratum 5 contain substance P immunoreactivity (yellow) or contact the substance P-IR processes. However, there are substance P-IR processes without immunoreactive gCCK and gCCK-IR processes without immunoreactive substance P. Stack = 5 × 0.5 µm. B,C: In peripheral retina, the colocalization of immunoreactive gCCK and substance P in somata was investigated in the GCL in a whole-mount preparation. Some of the gCCK-IR somata contained substance P-IR (B, arrow) and others did not (C, arrow). Stacks 8 × 0.5 µm. D: Stratum 5 of the IPL at a higher magnification is shown in a whole mount; only a subset of the gCCK-IR processes contain substance P immunoreactivity. Stack = 6 × 0.5 µm. E: In this single optical section (1 µm), the gCCK (red, left) and substance P immunoreactivity (green, middle) are shown separately and combined on the right. This clearly shows gCCK-IR processes that contain substance P immunoreactivity (arrows) and many processes that contain only immunoreactive gCCK or only immunoreactive substance P (arrowheads). Contacts (asterisks) between these processes were also detected. All scale bars = 10 µm.