Recoverin is a 23 kDa Ca2+binding protein that has been detected primarily in vertebrate photoreceptors. The role of recoverin in phototransduction has been investigated using a variety of biochemical methods. Initial reports suggesting that recoverin regulates photoreceptor guanylyl cyclase have not been confirmed. Instead, recoverin appears to determine the lifetime of lightstimulated phosphodiesterase activity, perhaps by regulating rhodopsin phosphorylation. Retinal recoverin is heterogeneously fatty acylated at its ammo-terminus. The amino-terminal fatty acid appears to be involved in the interaction of recoverin with photoreceptor membranes.

Human, macaque monkey, and rat retinas were immunostained with a polyclonal antibody preparation against purified recoverin, a 23-kD calcium-binding protein isolated from bovine retina that localizes to rods and cones (Dizhoor et al., 1991). In addition to immunoreactive photoreceptors, we have identified subpopulations of recoverin-positive bipolar cells in all three species. Results from immunostaining with progressive dilutions of anti-recoverin and preadsorption of the antibody with a dilution series of purified recoverin showed that photoreceptors and bipolar cells had similar affinities for the antibody and suggested that the molecule recognized by the antibody in both cell types is recoverin. Immunoreactivity for recoverin and protein kinase C, a selective marker for all rod bipolar cells, was found in separate bipolar cell populations. Recoverin immunoreactivity is therefore a characteristic of certain cone bipolar cell types.

In rat retina, anti-recoverin labeled two morphologically distinct subpopulations of cone bipolar cells whose axonal arbors stratified at different depths in the inner plexiform layer (IPL). The bipolar cells labeled with anti-recoverin did not correspond to those that were reactive for calbindin, another cone bipolar cell marker.

Human and monkey retinas also had two populations of cone bipolar cells that were recoverin-positive. One population showed a distinct pattern of narrow bistratification at the outer border of the IPL and a regular mosaic arrangement of its axonal arbors, suggesting that the entire population of a single cone bipolar type was labeled. Cell density, dendritic morphology, and axonal-field size and stratification indicate that anti-recoverin selectively stains the flat midget (presumed OFF-center) cone bipolar cell type observed previously in Golgi preparations. By contrast the second bipolar cell population had axonal stratification in the inner half of the IPL and showed an unusual but consistent morphology and spatial distribution. Individual cells were intensely stained but were present at an extremely low density (~2−5 cells/mm2). These cells had multibranched dendritic trees characteristic of the diffuse bipolar cell class, but very small axonal fields in the size range of the midget bipolar class. Neither of the two recoverin-positive bipolar cell types in monkey was labeled with anti-calbindin or anti-cholecystokinin. An antibody preparation against bovine pineal hydroxyindole-O-methyltransferase (HIOMT) labeled photoreceptors and bipolar cells that closely resembled the recoverin-positive bipolar cells in human and rat retinas. Preadsorption of this antibody preparation with purified recoverin abolished immunostaining of the bipolar cells, suggesting that the anti-HIOMT preparation contains antibodies against recoverin, which is known to be present in the bovine pineal gland.

Bipolar cells are not only important for visual processing but input from these cells may underlie the reorganization of ganglion cell dendrites in the inner plexiform layer (IPL) during development. Because little is known about the development of bipolar cells, here we have used immunocytochemical markers and dye labeling to identify and follow their differentiation in the neonatal ferret retina. Putative cone bipolar cells were immunoreacted for calbindin and recoverin, and rod bipolar cells were immunostained for protein kinase C (PKC). Our results show that calbindin-immunoreactive cone bipolar cells appear at postnatal day 15 (P15), at which time their axonal terminals are already localized to the inner half of the IPL. By contrast, recoverin-immunoreactive cells with terminals in the IPL are present at birth, but many of these cells may be immature photoreceptors. By the second postnatal week, recoverin-positive cells resembling cone bipolar cells were clearly present, and with increasing age, two distinct strata of immunolabeled processes occupied the IPL. PKC-containing rod bipolar cells emerged by the fourth postnatal week and at this age have stratified arbors in the inner IPL. The early bias of bipolar axonal arbors in terminating in the inner or outer half of the IPL is confirmed by dye labeling of cells with somata in the inner nuclear layer. At P10, several days before ribbon synapses have been previously observed in the ferret IPL, the axon terminals of all dye-labeled bipolar cells were clearly stratified. The results suggest that bipolar cells could provide spatially localized interactions that are suitable for guiding dendritic lamination in the inner retina.

The present study has examined the spatial and temporal expression patterns of various proteins associated with the structure and function of mature photoreceptor outer segments in the developing ferret's retina using immunocytochemistry and RT-PCR. One set of proteins, including rod opsin, arrestin, and recoverin, was detected progressively in photoreceptors as they became postmitotic, being expressed well before the differentiation of outer segments. A second set of proteins, including β- and γ-transducin, cGMP-phosphodiesterase, phosducin, rhodopsin kinase, rod cGMP-gated cation channel protein, and peripherin, displayed a contrasting temporal onset and pattern of spatial emergence. These latter proteins first became detectable either shortly before or coincident with outer segment formation, and were expressed simultaneously in both older and younger photoreceptor cells. A third set, the short wavelength-sensitive (SWS) and medium wavelength-sensitive (MWS) cone opsin proteins, was the last to be detected, but materialized in a spatio-temporal pattern reminiscent of the neurogenetic gradient of the cones. These different spatial and temporal patterns indicate that cellular maturation must play a primary role in regulating the onset of expression of some of these proteins, while extrinsic signals must act to coordinate the expression of other proteins across photoreceptors of different ages.

We investigated the synaptic connectivity of two populations of recoverin-labeled bipolar cells and GABAergic neurons in the inner plexiform layer (IPL) of the rat retina. Two types of cone bipolar cells, type 2 and type 8, were stained with anti-recoverin antibodies, and GABAergic neurons were stained with anti-glutamic acid decarboxylase (GAD) antibodies. Type 2 cone bipolar axons received synaptic input from amacrine cell processes in 177 cases; among these amacrine cell processes, 92 processes (52.0%) were GAD-like immunoreactive. A total of 159 amacrine cell processes, which are presynaptic to type 8 cone bipolar cells, were observed. Among these processes, 117 processes (73.6%) were GAD-like immunoreactive. The postsynaptic elements at the ribbon synapses of recoverin-labeled cone bipolar cells were observed in 482 processes. In both type 2 and type 8 cone bipolar cells, the major output was to amacrine cell processes. At the ribbon synapses of the type 2 cone bipolar cells, 224 of the postsynaptic profiles were amacrine cell processes, 97 processes (43.3%) were GAD-like immunoreactive. In type 8 cone bipolar cells, 45 processes (30.2%) of 149 amacrine cell processes were GAD-like immunoreactive. Our results provide morphological evidence that GABA is a major transmitter involved in the visual processing of type 2 and 8 cone bipolar cells and GABA may have a stronger influence on type 8 cone bipolar cells than type 2 cone bipolar cells in the IPL of the rat retina.