Mammalian Rod Research Articles

Cyclic-nucleotide-gated channels: pore topology in desensitizing E19A mutants.

In cyclic-nucleotide-gated "CNG" channels, the pore-loop "P-loop" is formed by the amino acid residues R345-S371 (here called R1-S27). Residue E19 determines the channel's interaction with extracellular divalent cations and contributes to ion conduction. Neutralization of this residue with alanine introduces channel desensitization. We have used serial cysteine mutagenesis to study P-loop topology in the alpha subunit of the mammalian rod CNG channels containing the E19A substitution. The pore topology was tested in the closed channel state and, when cGMP was present, during and after desensitization. With E19A substitution, the T15C, T16C, I17C and T20C mutants desensitized more slowly than controls. Moreover, the typical rundown produced by the I17C substitution in the wild-type "w.t." background was considerably reduced. Overall, with the E19A substitution, the accessibility pattern tested by applying the thiol-specific reagents Cd2+ and MTSET from the cytoplasmic side of the plasma membrane was similar to that observed with the w.t. Moreover, P22C channels were not inhibited by Cd2+ and MTSET (which do not cross the lipid bilayer) applied from the inside, but were blocked by MTSEA (which permeates the plasma membrane) also applied from the inside. This suggests that the residues following E19 remain accessible from the external side after E19A substitution. Thus, although the residues T15 to T20 seemed to participate in the structural rearrangements producing desensitization, no major P-loop remodelling occurs in desensitizing channels.

Voltage-activated Ca2+ channels and ionotropic GABA receptors localized at axon terminals of mammalian retinal bipolar cells.

A preparation of isolated presynaptic terminals of rat retinal rod bipolar cells was developed. Patch-clamp recordings were performed on the isolated terminal to determine the type(s) of voltage-activated Ca2+ channels and the contribution of GABA(A) and GABA(C) receptor-mediated currents localized in the terminal region. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents, with properties similar to those found in intact cell recordings, were observed in the isolated terminal recordings. Consistent with previous studies, the HVA Ca2+ currents are L-type since the currents were blocked by low micromolar concentrations of nimodipine and potentiated by BayK 8644. Also, both GABA(A) and GABA(C) receptor-mediated currents were observed in the isolated terminal. The current density of GABA(C) receptors in the terminal was more than three times higher than that in the soma. In contrast, the current density of GABA(A) currents between the terminal and the soma was not significantly different. Assessed by 100 microM GABA, the contributions of GABA(A) and GABA(C) receptors to the total GABA-mediated currents at the terminal were comparable. This study directly demonstrates the localization of LVA Ca2+ channels at the axon terminal of mammalian rod bipolar cells, suggesting that LVA Ca2+ channels may play a role in bipolar cell transmitter release. Results of this study also support the notion that both types of ionotropic GABA receptors regulate synaptic transmission in mammalian rod bipolar cells. In addition, this study reports for the first time the feasibility of direct patch-clamp recordings of isolated axon terminals of mammalian retinal bipolar cells. The isolated presynaptic terminal preparation of mammalian retinal bipolar cells could be a valuable system for the study of transmitter release in the central nervous system (CNS).

In vitro inhibition of antirecoverin immunoglobulin-mediated death of mammalian photoreceptor cells

Cancer-associated retinopathy (CAR) is a blinding disease, which can be mediated by autoimmune reactions with a specific calcium-binding retinal protein, recoverin. A number of recent studies demonstrate that agents that mobilize intracellular calcium can protect neurons from apoptotic death induced by a variety of insults. In this study, we investigated the effect of one such agent, potassium, on the survival of mammalian rod photoreceptors exposed to antirecoverin IgG. Primary cell cultures of rat retinal neurons were grown in a chemically defined medium, and cells were exposed to antirecoverin IgG for 72 hr in various concentrations of potassium and the surviving cells counted. Rod photoreceptors were quantitated using antirhodopsin immunofluorescence microscopy, and total cell numbers were determined by 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei. Apoptosis was evaluated by TdT-mediated biotin-dUTP nick-end labeling (TUNEL), cell death-detection ELISA, and DNA laddering. The present study shows that elevated extracellular K+ ([K+](o)) protects retinal neurons from antirecoverin antibody-mediated cell death. The protective effects of ([K+](o)) were shown to be time- and dose-dependent. The inhibition of antirecoverin IgG-mediated death of photoreceptors by elevated ([K+](o)) suggests that the mobilization of internal calcium stores rescues the cells by interfering with apoptotic signal transduction pathways. These data also suggest that the death of photoreceptor cells occurring in CAR possibly can be prevented by reagents and/or environmental changes that mobilize intracellular calcium.

A Fully Functional Rod Visual Pigment in a Blind Mammal: A CASE FOR ADAPTIVE FUNCTIONAL REORGANIZATION?

In the blind subterranean mole rat Spalax ehrenbergi superspecies complete ablation of the visual image-forming capability has been accompanied by an expansion of the bilateral projection from the retina to the suprachiasmatic nucleus. We have cloned the open reading frame of a visual pigment from Spalax that shows >90% homology with mammalian rod pigments. Baculovirus expression yields a membrane protein with all functional characteristics of a rod visual pigment (lambda(max) = 497 +/- 2 nm; pK(a) of meta I/meta II equilibrium = 6.5; rapid activation of transducin in the light). We not only provide evidence that this Spalax rod pigment is fully functional in vitro but also show that all requirements for a functional pigment are present in vivo. The physiological consequences of this unexpected finding are discussed. One attractive option is that during adaptation to a subterranean lifestyle, the visual system of this mammal has undergone mosaic reorganization, and the visual pigments have adapted to a function in circadian photoreception.

Salamander rods and cones contain distinct transducin alpha subunits.

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin a subunits (G alpha t) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of G alpha t. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander G alpha t-1 has 91.2-93.7% amino acid sequence identity to mammalian rod G alpha t subunits and 79.7-80.9% to mammalian cone Gats. The salamander G alpha t-2 has 86.2-87.9% sequence identity to mammalian cone G alpha ts and 78.9-80.9% to mammalian rod G alpha ts at the amino acid level. The G alpha t-1 cDNA encodes 350 amino acids while the G alpha t-2 cDNA encodes 354 residues, which is typical for rod and cone G alpha ts, respectively, and we thus identified the G alpha t- 1 as rod and G alpha t-2 as cone G alpha t. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone G alpha t subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod G alpha t and 2.6 kb for cone G alpha t. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod G alpha t is localized to rods, predominantly in the outer segments; similarly, cone G alpha t is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

Do Phosphatidylinositides Modulate Vertebrate Phototransduction?

Mammalian rod cyclic nucleotide gated (CNG) channels (i.e., alpha plus beta subunits) are strongly inhibited by phosphatidylinositol 4, 5-bisphosphate (PIP(2)) when they are expressed in Xenopus oocytes and studied in giant membrane patches. Cytoplasmic Mg-ATP inhibits CNG currents similarly, and monoclonal antibodies to PIP(2) reverse the effect and hyperactivate currents. When alpha subunits are expressed alone, PIP(2) inhibition is less strong; olfactory CNG channels are not inhibited. In giant patches from rod outer segments, inhibition by PIP(2) is intermediate. Other anionic lipids (e.g., phosphatidyl serine and phosphatidic acid), a phosphatidylinositol-specific phospholipase C, and full-length diacylglycerol have stimulatory effects. Although ATP also potently inhibits cGMP-activated currents in rod patches, the following findings indicate that ATP is used to transphosphorylate GMP, generated from cGMP, to GTP. First, a phosphodiesterase (PDE) inhibitor, Zaprinast, blocks inhibition by ATP. Second, inhibition can be rapidly reversed by exogenous regulator of G-protein signaling 9, suggesting G-protein activation by ATP. Third, the reversal of ATP effects is greatly slowed when cyclic inosine 5'-monophosphate is used to activate currents, as expected for slow inosine 5' triphosphate hydrolysis by G-proteins. Still, other results remain suggestive of regulatory roles for PIP(2). First, the cGMP concentration producing half-maximal CNG channel activity (K(1/2)) is decreased by PIP(2) antibody in the presence of PDE inhibitors. Second, the activation of PDE activity by several nucleotides, monitored electrophysiologically and biochemically, is reversed by PIP(2) antibody. Third, exogenous PIP(2) can enhance PDE activation by nucleotides.

8] Molecular structure of membrane-targeting calcium sensors in vision: Recoverin and guanylate cyclase-activating protein 2

Publisher Summary An important class of calcium-binding proteins present in retinal photoreceptor cells includes recoverin from mammalian rods, guanylate cyclase-activating proteins (GCAP-1, GCAP-2, and GCAP-3) from mammalian rods and cones, and guanylate cyclase inhibitory protein (GCIP) from frog rods. The recoverin branch of the EF-hand superfamily includes neuronal Ca 2+ sensors such as neurocalcin, frequenin, and hippocalcin. Mass spectrometric analysis of retinal recoverin and the GCAP proteins revealed that they are myristoylated at the amino terminus. GCAP-2 exhibits different membrane-targeting properties from those of recoverin. The myristoylated and unmyristoylated forms of GCAP-2 bind to membranes at low Ca 2+ concentrations ( 50 mM NaCl).This chapter presents a detailed structural analysis of recoverin and GCAP-2. The Ca 2+ -induced structural changes in these proteins are important for elucidating their membrane-targeting mechanisms and for understanding the molecular mechanism of Ca 2+ -sensitive regulation of phototransduction.

13] Electroretinographic determination of human Rod flash response in vivo

This chapter describes a “paired-flash” electroretinogram (ERG) method that circumvents the constraint just noted, and in human subjects yields approximate determination of the full time course of the massed rod response to a test flash of arbitrary intensity. Studies to date investigating the paired-flash technique in human subjects indicate that this method yields, to good approximation, the full time course of the massed rod photoreceptor response to a test flash. Support for this conclusion comes primarily from the correspondence of kinetic, sensitivity, and light adaptation properties of the paired-flash-derived response with photocurrent response properties of human and other mammalian rods in vitro . Results obtained with the paired-flash method motivate further work in both human subjects and experimental animals to refine this in vivo approach for studying rod phototransduction. The chapter also identifies a number of considerations likely to be important for future development of the technique, including the refinement of procedures to subtract the cone contribution at short interflash intervals, and to better correct for postreceptor contributions and desensitization of the probe flash response.

Minimal Domain Requirement for Cation Transport by the Potassium-dependent Na/Ca-K Exchanger: COMPARISON WITH AN NCKX PARALOG FROM CAENORHABDITIS ELEGANS

The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent (45)Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassium-dependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.

CGMP Binding to Noncatalytic Sites on Mammalian Rod Photoreceptor Phosphodiesterase Is Regulated by Binding of Its γ and δ Subunits

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, alpha beta gamma2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, alpha beta gamma2 delta) can release and bind cGMP at both noncatalytic sites; the delta subunit is likely responsible for this difference in cGMP exchange rates. Removal of the delta and/or gamma subunits yields a catalytic alphabeta dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (KD >1 microM); cGMP binding affinity at the second site (KD approximately 60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of gamma subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the gamma subunit with the alphabeta dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.

Antioxidant Activity of Retinol, Glutathione, and Taurine in Bovine Photoreceptor Cell Membranes

The antioxidant activities of compounds endogenous to mammalian rod outer segments (ROS) were investigated in vitro by measuring the oxidative loss of polyunsaturated fatty acids (PUFA's) from the membranes of intact ROS and from liposomes made from ROS phospholipids (PL) to which lipid soluble compounds had been added. The membranes were exposed to the water-soluble oxidant 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). Retinol protected PUFA's in ROS liposome PL's, whereas retinaldehyde promoted lipid peroxidation. When isolated ROS were stimulated to produce endogenous retinol, PUFA loss was inhibited by up to 17%. These findings suggest an antioxidant function for the enzymatic reduction of retinaldehyde to retinol during the visual cycle. Water-soluble antioxidants, taurine and reduced glutathione (GSH), were investigated individually and in combination with retinol in ROS PL liposomes. GSH protected PUFA's in ROS PL liposomes. Taurine alone showed little antioxidant activity, but in combination with retinol it protected lipids twice as much as retinol alone. These results support previous findings that taurine protects ROS lipids during exposure to cyclic light.

Crystal structure of recombinant bovine neurocalcin.

The crystal structure of calcium-bound unmyristoylated bovine neurocalcin from Escherichia coli has been determined at 2.4 A resolution. The three-dimensional structure reveals a highly compact structure consisting of: (i) two pairs of calcium-binding EF-hands (EF1-EF2 and EF3-EF4); (ii) a calcium ion bound at EF2, EF3 and EF4 sites; and (iii) an EF1-hand that is disabled from calcium-binding due to a Cys-Pro sequence in the Ca2+-binding loop. The crystal structure of neurocalcin resembles photoreceptor recoverin in overall topology, however its EF2- and EF4-hands differ. Recently, neurocalcin in the calcium-bound state has been shown to stimulate mammalian rod outer segment membrane guanylate cyclase. A possible site for cyclase activity based on the three-dimensional structure is discussed.

Rod photoreceptor maturation does not vary with retinal eccentricity in mammalian retina

Purpose. Test the hypothesis that the development of mammalian rod outer segments (ROS) varies with retinal eccentricity. Methods. During the period of photoreceptor cell development, ROS lengths, opsin mRNA and (rhod)opsin were measured in central and peripheral retina of cows and pigmented rats. Published ROS length and/or rhodopsin data from albino rats, cows and monkeys were re-analyzed. Logistic growth curves were fitted to the newly obtained and published data. Within a species, growth in central and peripheral regions was compared. Results. The logistic growth curves fit all the data well and provide an excellent view of the developmental increases in ROS length, opsin mRNA and (rhod)opsin in each retinal region. Within a species, the growth curves for ROS length, opsin mRNA and (rhod)opsin concentration are superimposable. The age at which ROS length reaches 50% of its adult value is invariant with eccentricity. An exception to this pattern is the simian parafoveal ROS, which appears to have a delayed course of development. Conclusions. The hypothesis is disproved. Unlike rod photoreceptor cell genesis, ROS development is invariant with retinal eccentricity. Primate parafoveal ROS appear to have a different pattern of development.

Transmitter concentration at a three-dimensional synapse.

Transmitter concentration at a three-dimensional synapse. J. Neurophysiol. 80: 3163-3172, 1998. At intensities from starlight to 1000-fold brighter, the mammalian rod synapse transmits a binary signal, the capture of 0 or 1 photon. Zero is signified by tonic exocytosis, and 1 is signified by a brief pause. The synapse is three dimensional: vesicles discharge at the apex of a deep cleft created by the invagination of four postsynaptic processes. Two horizontal cell spines bearing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors reach near to the release sites (16 nm), and two bipolar dendrites bearing mGluR6 receptors end far from the release sites (up to 640 nm). We considered two hypotheses for signal transfer: transmitter quanta might be integrated in the cleft and sensed as a steady concentration (high for 0 and low for 1); or quanta might be sensed at the postsynaptic membrane as discrete postsynaptic potentials (PSPs) and integrated within the dendrite. We calculate from a passive diffusion model that the invagination empties rapidly (tau approximately 1.7 ms). Further calculations suggest that a glutamate concentration high enough to hold a bipolar cell in darkness at one end of its response range would require approximately 4,000 vesicles/s. On the other hand, the glutamate pulse from a single vesicle would reach both nearby AMPA receptors (low affinity) and distant mGluR6 receptors (high affinity) at spatiotemporal concentrations matched to their apparent binding affinities. Thus one vesicle could evoke a discrete PSP in all four postsynaptic processes. We calculate from a stochastic model that PSPs could transfer the binary signal at approximately 100 vesicles/s. Thus dendritic integration of unitary PSPs is both plausible and 40-fold more efficient than the alternative mechanism. The rod's deep invagination, rather than serving to pool transmitter, may serve to prevent "spillover" of transmitter to neighboring rods. Spillover, by pooling the noise from neighboring rods, would impair transmission of their binary signals.

A Novel Signaling Pathway from Rod Photoreceptors to Ganglion Cells in Mammalian Retina

Current understanding suggests that mammalian rod photoreceptors connect only to an ON-type bipolar cell. This rod-specific bipolar cell excites the AII amacrine cell, which makes connections to cone-specific bipolar cells of both ON and OFF type; these, in turn, synapse with ganglion cells. Recent work on rabbit retina has shown that rod signals can also reach ganglion cells without passing through the rod bipolar cell. This route was thought to be provided by electrical gap junctions, through which rods signal directly to cones and thence to cone bipolar cells. Here, we show that the mouse retina also provides a rod pathway bypassing the rod bipolar cell, suggesting that this is a common feature in mammals. However, this alternative pathway does not require cone photoreceptors; it is perfectly intact in a transgenic mouse whose retina lacks cones. Instead, the results can be explained if rods connect directly to OFF bipolar cells.

GABAA and GABAC receptors on mammalian rod bipolar cells.

Rod bipolar (RB) cells of mammalian retinae receive synapses from different gamma-aminobutyric acid (GABAergic) amacrine cells in the inner plexiform layer (IPL). We addressed the question whether RB cells of the rabbit and of the rat retina express different types of GABA receptors at these synapses. RB cells were immunolabeled in vertical sections of rat retinae with an antibody against protein kinase C (PKC). The sections were double-labeled for the alpha 1, alpha 2, alpha 3, or gamma 2 subunits of the GABAA receptor. Punctate immunofluorescence, which represents synaptic localization, was found for all four subunits. Many of the alpha 1-, alpha 3-, or gamma 2-immunoreactive puncta coincided with the axon terminals of the PKC-immunolabeled RB cells. Sections and wholemounts of rabbit retinae were also double labeled for PKC and the rho subunits of the GABAC receptor. Rabbit RB cells were decorated by many rho-immunoreactive puncta, which were shown by electron microscopy to represent synaptic localization. Previous work from our laboratory has shown that the alpha 1, alpha 2, alpha 3, and rho subunits are not found within the same synapse but are expressed at different synaptic sites. Taken together, these results suggest that RB cells of mammalian retinae express at least three different types of GABA receptors at synaptic sites in the IPL: GABAC receptors, GABAA receptors containing the alpha 1 subunit, and GABAA receptors containing the alpha 3 subunit.

Noise removal at the rod synapse of mammalian retina.

Mammalian rods respond to single photons with a hyperpolarization of about 1 mV which is accompanied by continuous noise. Since the mammalian rod bipolar cell collects signals from 20-100 rods, the noise from the converging rods would overwhelm the single-photon signal from one rod at scotopic intensities (starlight) if the bipolar cell summed signals linearly (Baylor et al., 1984). However, it is known that at scotopic intensities the retina preserves single-photon responses (Barlow et al., 1971; Mastronarde, 1983). To explore noise summation in the rod bipolar pathway, we simulated an array of rods synaptically connected to a rod bipolar cell using a compartmental model. The performance of the circuit was evaluated with a discriminator measuring errors in photon detection as false positives and false negatives, which were compared to physiologically and psychophysically measured error rates. When only one rod was connected to the rod bipolar, a Poisson rate of 80 vesicles/s was necessary for reliable transmission of the single-photon signal. When 25 rods converged through a linear synapse the noise caused an unacceptably high false positive rate, even when either dark continuous noise or synaptic noise where completely removed. We propose that a threshold nonlinearity is provided by the mGluR6 receptor in the rod bipolar dendrite (Shiells & Falk, 1994) to yield a synapse with a noise removing mechanism. With the threshold nonlinearity the synapse removed most of the noise. These results suggest that a threshold provided by the mGluR6 receptor in the rod bipolar cell is necessary for proper functioning of the retina at scotopic intensities and that the metabotropic domains in the rod bipolar are distinct. Such a nonlinear threshold could also reduce synaptic noise for cortical circuits in which sparse signals converge.

High expression levels in cones of RGS9, the predominant GTPase accelerating protein of rods.

RGS9 is a member of the RGS family of GTPase accelerating proteins (GAPs) for heterotrimeric G proteins. We have explored its contribution to GTPase acceleration in mammalian rod and cone photoreceptors. When RGS9 was specifically removed from detergent extracts of rod outer segments by immunodepletion, the extracts lost nearly all of their GAP activity stimulatable by the inhibitory subunit of cGMP phosphodiesterase. Immunolocalization using monoclonal antibodies and confocal microscopy revealed that RGS9 is present in cones at significantly higher levels than in rods. Thus, RGS9 is the predominant source of GAP activity in rod outer segments, and RGS9 concentration emerges as a potentially important determinant of the faster response kinetics and lower sensitivity of mammalian cones, as compared with rods.

Role of the low-affinity NGF receptor (p75) in survival of retinal bipolar cells.

We have examined the role of neurotrophins in promoting survival of mammalian rod bipolar cells (RBC) in culture. Retinas taken from 8- to 10-day-old Long-Evans rats were dissociated and cultured in media supplemented with either nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (FGF-2). Survival was measured by the number of cells that were immunoreactive for alpha-, beta-, gamma-PKC, a bipolar cell-specific marker. Compared to untreated cultures, CNTF had no effect on RBC survival, while NGF and NT-3 increased survival only slightly. BDNF, however, increased survival by approximately 300%. Similar results were obtained with FGF-2. Both nerve growth factor (NGF) and an antibody (anti-REX) which interferes with binding to the 75-kD low-affinity neurotrophin receptor (p75NTR) eliminated BDNF-promoted survival, but had no effect on FGF-2-mediated survival. Interestingly, p75NTR was expressed by retinal glia (Müller cells), but not by the bipolar cells themselves, providing for the possibility that BDNF might induce Müller cells to produce a secondary factor, perhaps FGF-2, which directly rescues RBCs. In support of this hypothesis, an antibody that neutralizes FGF-2 attenuated the trophic effects of BDNF, and dramatically reduced survival in cultures with no added growth factors, indicating that there may be an endogenous source of FGF-2 that promotes survival of RBCs in culture. We suggest that BDNF increases production or release of FGF-2 by binding to p75NTR on Müller cells.