Differential degeneration of rod/cone bipolar cells during retinal degeneration in Royal College of Surgeons rats

Dear Editor,Royal college of surgeons(RCS)rat is an inherited retinal degeneration rat caused by mutation of Mertk in the retinal pigment epithelial(RPE)cells^[1].In this dystrophic rat,the dysfunction of RPEs leads to the progressive death of photoreceptors,with rods initially affected.Many studies have attempt to transplant various stem cells.     

Neurochemical heterogeneity of retinal bipolar cells.

BACKGROUND: Retinal bipolar cells show neurochemical changes in response to retinal insult, which could have profound effects on retinal function or recovery after insult however, the neurochemistry of bipolar cells is incompletely understood. METHODS The distribution of the amino acid neurotransmitters glutamate, gamma-aminobutyric acid (GABA), and glycine, and the metabolic amino acids, aspartate and glutamine, was examined immunocytochemically in the retina of the tiger salamander, a major model for the study of retinal anatomy and function. RESULTS: Each amino acid showed a unique distribution among retinal bipolar cells. All bipolar cells appear to contain glutamate and, under appropriate conditions, may also contain aspartate. GABA and glycine were restricted to a subset of conventionally placed bipolar cells located in the inner nuclear layer, but were not present in displaced bipolar cells located in the outer nuclear layer. In contrast, high levels of glutamine were almost exclusively restricted to displaced bipolar cells. Analysis of co-localization patterns revealed several different amino acid signatures within the bipolar cell population. CONCLUSIONS: The striking neurochemical heterogeneity among bipolar cells suggests important differences in signaling and metabolism that could affect cell survival, plasticity, and functional recovery from retinal insult in a cell-type-specific manner.     

Cone bipolar cells and cone synapses in the primate retina

Primate retinal bipolar cells synapsing with two adjacent cones (2C bipolars) are further described. Their synaptic contacts are either as the central (invaginating) component of the cone triads or as basal (flat) contacts on the membrane of the cone pedicle base. Correspondingly, their axons end either in the b (inner half or in the a (outer) half of the inner plexiform layer. The shape and size of the axon terminals of 2C bipolars are indistinguishable from those of adjacent midget bipolars. Therefore 2C bipolars, like midget bipolars, probably synapse with midget ganglion cells. Two C bipolars have not been identified as connected to foveal cones. But they are not restricted to the retinal periphery, as has previously been supposed, since they occur, mixed with midget (single cone) bipolars, throughout all parts of the retina from about 2.5 mm to at least 10.0 mm from the fovea. It is likely that 2C bipolars are a variant of the midget bipolars; and that they contact some members of the same population of cones, instead of the midgets. This paper briefly reviews, and raises some new, problems concerning our current understanding of the synaptic connectivity patterns of the midget, 2C, and diffuse cone bipolar cells.     

Retinal bipolar cells: temporal filtering of signals from cone photoreceptors

The temporal dynamics of the response of neurons in the outer retina were investigated by intracellular recording from cones, bipolar, and horizontal cells in the intact, light-adapted retina of the tiger salamander (Ambystoma tigrinum), with special emphasis on comparing the two major classes of bipolars cells, the ON depolarizing bipolars (Bd) and the OFF hyperpolarizing bipolars (Bh). Transfer functions were computed from impulse responses evoked by a brief light flash on a steady background of 20 cd/m(2). Phase delays ranged from about 89 ms for cones to 170 ms for Bd cells, yielding delays relative to that of cones of about 49 ms for Bh cells and 81 ms for Bd cells. The difference between Bd and Bh cells, which may be due to a delay introduced by the second messenger G-protein pathway unique to Bd cells, was further quantified by latency measurements and responses to white noise. The amplitude transfer functions of the outer retinal neurons varied with light adaptation in qualitative agreement with results for other vertebrates and human vision. The transfer functions at 20 cd/m(2) were predominantly low pass with 10-fold attenuation at about 13, 14, 9.1, and 7.7 Hz for cones, horizontal, Bh, and Bd cells, respectively. The transfer function from the cone voltage to the bipolar voltage response, as computed from the above measurements, was low pass and approximated by a cascade of three low pass RC filters ("leaky integrators"). These results for cone-->bipolar transmission are surprisingly similar to recent results for rod-->bipolar transmission in salamander slice preparations. These and other findings suggest that the rate of vesicle replenishment rather than the rate of release may be a common factor shaping synaptic signal transmission from rods and cones to bipolar cells.     

Synaptic contact between melanopsin-containing retinal ganglion cells and rod bipolar cells

PURPOSE: Evidence indicates that the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) receive input from rods and cones, which are thought to modulate the irradiance detecting system driving entrainment of the circadian system and pupillomotor control. This study was performed to identify retinal cells that have synaptic contact with ipRGCs. METHODS: Immunohistochemistry and high-power confocal microscopy were used to generate stacks of digital images of sections stained with antibodies against melanopsin, protein kinase C (PKCalpha), tyrosine hydroxylase (TH), presynaptic terminal markers (C-terminal binding protein 2 [CtBP2], vesicular monoamine transporter 2 [VMAT2] and postsynaptic marker (glutamate receptor subunit 4 [GluR4]). Results were analyzed in a computer-based three-dimensional reconstruction program for cellular contacts. RESULTS: Markers and melanopsin rod bipolar processes were found to have axosomatic and axodendritic contact with melanopsin-containing RGCs. Typically, three to four contacts were found on the soma of the melanopsin-containing RGCs, together with contacts on proximal dendrites. Contacts visualized by only CtBP2 immunoreactivity could also be demonstrated on melanopsin cell bodies and processes representing contacts with other types of bipolar cells. At the border of the inner plexiform layer (IPL) and inner nuclear layer (INL), where melanopsin processes stratify, contacts between melanopsin and TH or VMAT2 immunoreactivity processes were observed. CONCLUSIONS: Through confocal microscopy and computer-based three-dimensional analyses, this study demonstrates that melanopsin-containing RGCs have synaptic contact with PKC/CtBP2-containing rod bipolar cells and TH/VMAT2-immunoreactive amacrine cells through axodendritic and axosomatic contact, supporting electrophysiological observations that rods and cones signal to the melanopsin-containing intrinsically photosensitive RGCs.     

Rabbit cone bipolar cells: correlation of their morphologies with whole-cell recordings

The superfused retinal slice preparation was used to examine the morphology and glutamate-activated whole-cell currents of rabbit bipolar cells. There were six morphologically distinct types of cone bipolar cells and a rod bipolar cell that had axon terminals stratifying in stratum 3 to 5 of sublamina-b. All of these bipolar cell types exhibited an outward current in response to the application of the metabotropic glutamate receptor, mGluR6, agonist AP-4 (APB), and had I/V curves indicative of membrane channel closure. Conversely, there were no currents activated during the application of kainate, the AMPA/kainate receptor agonist. These data demonstrate they were on-bipolar cells. In addition, there were six morphologically distinct cone bipolar cells that stratified in sublamina-a. Every cell with axonal arborizations in stratum 1 and 2 exhibited an inward current when the ionotropic glutamate receptor agonist kainate was applied. This current was blocked by application of the AMPA/kainate receptor antagonist CNQX. These cells also decreased their membrane resistance in response to kainate, a characteristic of the opening of channels within the plasma membrane. Without exception, no cells stratifying in sublamina-a responded to the mGluR6 agonist AP-4, further identifying them as off-bipolar cells.     

The unitary event amplitude of mouse retinal on-cone bipolar cells

Light-evoked synaptic currents were recorded from on-cone bipolar cells in the mouse retina. Fluctuations in the synaptic current observed during maintained light steps were analyzed in order to estimate the amplitude of the underlying unitary event. The maximal synaptic current variance was 5-fold larger than the maximum expected from fluctuations in the number of active postsynaptic channels. Due to uncertainty in the contribution from channel variance, we calculated a range of values for the unitary event amplitude. The observed variance could be accounted for if 30-39 synaptic sites randomly generated unitary events with a waveform identical to the flash-response, and an amplitude of -3.1 to -2.4 pA. The amplitude is consistent with gating about five mGluR6 channels. The shape of the variance-mean relation suggests that in bright light transmitter release approaches zero, while in darkness transmitter release saturates the postsynaptic response. Thus the on-cone bipolar cell synapse is operating over its entire possible range. If it is assumed that the postsynaptic response saturates when one unitary event occurs per integration time, then a lower bound for the unitary event rate is 18 events/s/synaptic site. If the unitary event is generated by a single synaptic vesicle, the results suggest the total vesicle cycling rate available for encoding the on-cone bipolar cell signal is about 540-700 s(-1).     

GABA responses of rod bipolar cells in rabbit retinal slices

GABAergic responses of rabbit rod bipolar cells were reexamined by using whole-cell recordings in the superfused slice preparation to determine if there is GABA(C) receptor input to their axon terminal and to characterize the contribution that GABA(A) and GABA(C) receptors make to the total GABA current on the axon terminals of these cells. Pharmacological agents specifically blocking GABA(A) and GABA(C) receptor currents demonstrated that 37% of the GABA-activated current was blocked by either the GABA(A) antagonists bicuculline or SR-95531, whereas the remaining 63% of the GABA current was blocked by a mixture of bicuculline and the GABA(C) antagonist TPMPA. This indicated that GABA(C) receptors were present on the axon terminal of the rabbit rod bipolar cell and that they were responsible for mediating the bicuculline insensitive GABA current.     

Center surround receptive field structure of cone bipolar cells in primate retina

In non-mammalian vertebrates, retinal bipolar cells show center-surround receptive field organization. In mammals, recordings from bipolar cells are rare and have not revealed a clear surround. Here we report center-surround receptive fields of identified cone bipolar cells in the macaque monkey retina. In the peripheral retina, cone bipolar cell nuclei were labeled in vitro with diamidino-phenylindole (DAPI), targeted for recording under microscopic control, and anatomically identified by intracellular staining. Identified cells included 'diffuse' bipolar cells, which contact multiple cones, and 'midget' bipolar cells, which contact a single cone. Responses to flickering spots and annuli revealed a clear surround: both hyperpolarizing (OFF) and depolarizing (ON) cells responded with reversed polarity to annular stimuli. Center and surround dimensions were calculated for 12 bipolar cells from the spatial frequency response to drifting, sinusoidal luminance modulated gratings. The frequency response was bandpass and well fit by a difference of Gaussians receptive field model. Center diameters were all two to three times larger than known dendritic tree diameters for both diffuse and midget bipolar cells in the retinal periphery. In one instance intracellular staining revealed tracer spread between a recorded cell and its nearest neighbors, suggesting that homotypic electrical coupling may contribute to receptive field center size. Surrounds were around ten times larger in diameter than centers and in most cases the ratio of center to surround strength was approximately 1. We suggest that the center-surround receptive fields of the major primate ganglion cell types are established at the bipolar cell, probably by the circuitry of the outer retina.     

Glycine- and GABA-activated inhibitory currents on axon terminals of rabbit cone bipolar cells

Glycine- and GABA-activated currents were examined in the axon terminals of 12 types of rabbit cone bipolar cells. In the superfused retinal slice, a cell was voltage clamped at 0 mV in the presence of cobalt; then glycine or GABA was puffed onto the axon terminal. Types CBa1, CBa2, and a few CBa1-2 cells demonstrated larger glycine-activated currents than GABA-activated ones. However, some OFF cells (CBa2(n), CBa1-2(n), CBa1(w)), most CBa1-2, and most ON cells (CBb3, CBb3-4, CBb3(n), and CBb4) displayed larger GABA-activated currents. The ON cell, CBb5, possessed only a GABA-activated current. The predominance of glycinergic currents in CBa1, CBa2, and a few CBa1-2 cells suggests a major input from the glycinergic AII amacrine cell and thus a key role for these cells in the rod bipolar pathway. Certain OFF cells (most CBa1-2) expressed larger GABA-activated currents. All types expressed both GABA(A) and GABAC currents about equally, although most OFF types (CBa1, CB a2(n), CBa1-2, and CBa2(n)) displayed a slightly greater GABA(A) component.     

Wide-field cone bipolar cells and the blue-ON pathway to color-coded ganglion cells in rabbit retina

Wide-field cone bipolar cells with sparse dendritic branching and proposed connectivity to blue cones were first identified in rabbit and cat. In rabbit, these were subdivided into type a (wa) and type b (wb), with axonal branching in sublamina a, and sublamina b, respectively, of the inner plexiform layer (IPL). Recent studies in rabbit support the earlier hypothesis of exclusive blue/short wavelength cone connectivity for both types. The homologues of wb cells (but not wa cells) have been identified in other mammals. The axonal branching of wa cone bipolar cells is shown to co-stratify with the dendrites of the "fiducial," type a starburst amacrine cell, although a few branches extend into sublamina b. The axon terminal of wb cone bipolar cells is shown to be narrowly stratified in stratum 5alpha, deep to the dendrites of the type b starburst amacrine cell. Rabbit ganglion cells postsynaptic to wa cells are unknown, but may include class III.2a cells, similarly stratified in the IPL. The wb axon terminal is shown here to co-stratify with and to make close, likely synaptic, contacts with the dendrites of a recently described morphological subtype of class II ganglion cell in rabbit retina, IIb2. Recent morpho-physiological correlation indicates that class IIb2 cells correspond to the blue-ON-center-X or ON-brisk-sustained ganglion cells, defined physiologically in rabbit. In contrast, the wb cell in cat retina must innervate the physiologically identified blue-ON-center-sluggish-sustained ganglion cell. In monkey retina, the wb-like bipolar cells apparently innervate a small, partly bi-stratified ganglion cell. Mammals share a common pathway from short-wavelength-sensitive (S/blue) cone photoreceptors to ON-center ganglion cells in sublamina b of the IPL, in the form of wb or wb-like cone bipolar cells, but the type of ganglion cell innervated appears to be particular, and may serve different functional roles in different mammalian orders.     

Rod and cone inputs to bipolar and horizontal cells of the Xenopus retina

This report summarizes some recent studies of the Xenopus retina in which intracellular recordings were made from photoreceptors, horizontal and bipolar cells. The studied cells were identified by injection of Lucifer yellow. Rod spectral sensitivity functions conformed to the density spectrum of a 524 nm pigment, those of cones to that of a 612 nm pigment. Horizontal cell responses reflected both these classes of photoreceptor input. Rod input evoked a slow waveform, with Vmax less than or equal to 18 mV, cone input a faster waveform with Vmax = 30-40 mV. In the mesopic state the horizontal response reflected both waveforms. Rod and cone inputs to the horizontal cells appeared not to act independently, in that a steady weak green background greatly enhanced the response to a superimposed red flash, but not the reverse. A third photoreceptor type (blue-sensitive rod, Y lambda max = 445 nm) provided input to a chromatic bipolar cell which was hyperpolarized by blue light and depolarized by red light. Such chromatic bipolars had broad areas of spatial integration and lacked center-surround organization.     

Differential expression of K+ currents in mammalian retinal bipolar cells

Whole-cell voltage-clamp recordings were performed to investigate voltage-dependent K+ currents in acutely isolated retinal cone bipolar cells (CBCs) from the rat. The physiological and pharmacological properties of the currents were compared with those in rod bipolar cells (RBCs). The K+ currents were found to be much larger in CBC than in RBCs. In addition, the currents in CBCs were activated and inactivated at more negative potentials. Based on the apparent inactivation property of the currents, CBCs were found to fall into two groups of cells that differed in the inactivation kinetics of IK(V) but did not correlate to the ON- and OFF-type. The IK(V) for the group of CBCs showing faster inactivation, as well as for all RBCs, contained two components with decay time constants around 0.1 and 1 s. The IK(V) for the group of CBCs showing slower inactivation only contained the slower component. Furthermore, three components of IK(V) were observed based on tetraethylammonium (TEA) sensitivity: high-sensitive, low-sensitive, and resistant component. The IK(V) for a portion of CBCs showing faster inactivation, as well as for all RBCs, contained all three components. The IK(V) for the remaining CBCs, including all of those CBCs showing slower inactivation, only contained the latter two components. This study reveals a differential expression of K+ currents in rat retinal bipolar cells, suggesting that K+ channels may play an important role in bipolar cell processing in mammalian retinas.     

Rod and cone contribution to adaptation processes in cat retinal ganglion cells

Extracellular ganglion cell responses were recorded to investigate mechanisms of light adaptation. Monochromatic test spots (575 nm) were projected onto the receptive field center of off-center cells and superimposed on a steady blue-green Ganzfeld background (Schott Filter BG 28), the strength of which was increased in steps of 0.5 log units to adapt rods. Response vs. log intensity functions were determined over a range of 7 log units of test light irradiance at each background level. At higher adaptation levels response thresholds followed the typical Weber function. Surprisingly at lower adaptation levels the sensitivity of the cell increased by about 0.7 log units, most markedly in a range of 1 log unit of moderate light adaptation when the background was changed from dark to the dimmest detectable background (10^–5lm/m²). In the dark-adapted state a small off-response of long latency (40–100ms at 10² quanta · s^–1 · m^–2) is observed at low rod stimulating test light irradiances. A transition to a cone-dominated transient response of 2 to 5 ms duration occurred at high intensities (10⁵ quanta · s^–1 · m²). At mesopic levels the two responses seem to cancel each other, rendering a delayed off-response that is probably the result of rod-cone interaction. As in psychophysics, saturation can be observed at very high background intensities (10⁶ quanta · s^–1 m^–2). These data suggest interactions between rods and cones that determine the sensitivity of cat retinal ganglion cells at low levels of adaptation for suprathreshold stimuli.     

Contrast encoding in retinal bipolar cells: current vs. voltage

To investigate the influence of voltage-sensitive conductances in shaping light-evoked responses of retinal bipolar cells, whole-cell recordings were made in the slice preparation of the tiger salamander, Ambystoma tigrinum. To study contrast encoding, the retina was stimulated with 0.5-s steps of negative and positive contrasts of variable magnitude. In the main, responses recorded under voltage- and current-clamp modes were remarkably similar. In general agreement with past results in the intact retina, the contrast/response curves were relatively steep for small contrasts, thus showing high contrast gain; the dynamic range was narrow, and responses tended to saturate at relatively small contrasts. For ON and OFF cells, linear regression analysis showed that the current response accounted for 83-93% of the variance of the voltage response. Analysis of specific parameters of the contrast/response curve showed that contrast gain was marginally higher for voltage than current in three of four cases, while no significant differences were found for half-maximal contrast (C50), dynamic range, or contrast dominance. In sum, the overall similarity between current and voltage responses indicates that voltage-sensitive conductances do not play a major role in determining the shape of the bipolar cell's contrast response in the light-adapted retina. The salient characteristics of the contrast response of bipolars apparently arise between the level of the cone voltage response and the postsynaptic current of bipolar cells, via the transformation between cone voltage and transmitter release and/or via the interaction between the neurotransmitter glutamate and its postsynaptic receptors on bipolar cells.     

Suppression of voltage-dependent K+ currents in retinal bipolar cells by ascorbate

Ascorbate, often used as an antioxidant in neural studies, may also serve as a neuromodulator in the vertebrate central nervous system (CNS), in that it modulates the synaptic actions of glutamate and dopamine. Retina of fish contain a high concentration of ascorbate. The release and/or uptake of neurotransmitters are related to membrane potential, which to a large extent is determined by the activity of K+ channels. As retinal bipolar cells are subject to synaptic input from glutamatergic and dopaminergic sources, the effects of ascorbate on voltage-dependent K+ currents (I(K)(v)) of the mixed rod-cone ON-center bipolar cells (Mb) in goldfish retinal slices were studied using whole-cell recording techniques. I(K)(V) was suppressed reversibly 60% by 100-200 microM ascorbate. The effect of ascorbate was not due to changes in pH, oxidative stress, lipid peroxidation, any Ca2+-dependent or Na+-dependent action. However, the suppressive effect of ascorbate was blocked by cholera toxin and Wiptide, a protein kinase A (PKA) inhibitor. It is concluded that ascorbate, at physiological concentrations, inhibits I(K)(V) of bipolar cells via a Gs-protein-PKA system. This effect of ascorbate should be taken into account when using ascorbate as an antioxidant in retinal studies involving dopamine.