Responses Of Rod Bipolar Cells Research Articles

The Effect of PKCα on the Light Response of Rod Bipolar Cells in the Mouse Retina.

Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse. Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice. Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration. Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway.

Features of the Human Rod Bipolar Cell ERG Response During Fusion of Scotopic Flicker

The ability of the eye to distinguish between intermittently presented flash stimuli is a measure of the temporal resolution of vision. The aim of this study was to examine the relationship between the features of the human rod bipolar cell response (as measured from the scotopic ERG b-wave) and the psychophysically measured critical fusion frequency (CFF). Stimuli consisted of dim (-0.04 Td x s), blue flashes presented either singly, or as flash pairs (at a range of time separations, between 5 and 300 ms). Single flashes of double intensity (-0.08 Td x s) were also presented as a reference. Visual responses to flash pairs were measured via (1) recording of the ERG b-wave, and (2) threshold determinations of the CFF using a two-alternative forced-choice method (flicker vs. fused illumination). The results of this experiment suggest that b-wave responses to flash pairs separated by < 100 ms are electrophysiologically similar to those obtained with single flashes of double intensity. Psychophysically, the percepts of flash pairs < 100 ms apart appeared fused. In conclusion, the visual system's ability to discriminate between scotopic stimuli may be determined by the response characteristics of the rod bipolar cell, or perhaps by the rod photoreceptor itself.

Carbonic anhydrase‐related protein VIII is expressed in rod bipolar cells and alters signaling at the rod bipolar to AII‐amacrine cell synapse in the mammalian retina

Mutation of the gene encoding carbonic anhydrase-related protein VIII (CAVIII) results in motor coordination deficits in mice and humans, due to loss of this protein in Purkinje cells of the cerebellum. Recent studies have indicated that the CAVIII gene, Car8, is also expressed in rod bipolar cells (RBCs), a critical glutamatergic neuron for scotopic vision. We investigated the localization of CAVIII in the mouse and macaque retina, and utilized the wdl mouse, which has a null mutation in the Car8 gene, to determine how the loss of CAVIII affects retinal signaling. CAVIII immunoreactivity was observed in RBCs, with particularly high staining intensity in the axon terminals. In addition, weaker staining was observed in a subset of cone bipolar cells and γ-aminobutyric acid (GABA)ergic amacrine cells. Light-evoked current and voltage responses of RBCs were not altered in the wdl mutant. However, light-evoked current responses from the AII-amacrine cell, a postsynaptic partner at the RBC ribbon synapse, were significantly larger, and more prolonged than in control mice. These changes could not be attributed to alterations in calcium current activation or inactivation, or to changes in the density of RBCs. Furthermore, no gross synaptic alterations were evident in the wdl mutant at the light or ultrastructural level. These data provide evidence that the CAVIII protein, which is highly conserved in vertebrates, is selectively expressed within neural circuits, and may be important for modulating retinal neurotransmission.

PKCα Is Essential for the Proper Activation and Termination of Rod Bipolar Cell Response

Protein kinase (PKC)-α is abundant in retinal bipolar cells. This study was performed to explore its role in visual processing. PKCα-knockout (Prkca(-/-)) mice and control animals were examined by using electroretinography (ERG), light microscopy, and immunocytochemistry. The Prkca(-/-) mice showed no signs of retinal degeneration up to 12 months of age, but ERG measurements indicated a decelerated increase in the ascending limb of the scotopic (rod-sensitive) b-wave as well as a delayed return to baseline. These results suggest that PKCα is an important modulator that affects bipolar cell signal transduction and termination. Confocal microscopy of retinal sections showed that PKCα co-localized with calbindin, which indicates a PKCα localization in close proximity to the horizontal cell terminals. In addition, the implicit time of the ERG c-wave originating from the retinal pigment epithelium (RPE) and the recovery of photoreceptors from bleaching conditions were substantially faster in the knockout mice than in the wild-type control animals. These results suggest that PKCα is a modulator of rod-bipolar cell function by accelerating glutamate-driven signal transduction and termination. This modulation is of importance in the switch between scotopic and photopic vision. Furthermore, PKCα seems to play a role in RPE function.

SV2 Acts via Presynaptic Calcium to Regulate Neurotransmitter Release

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

A fast rod photoreceptor signaling pathway in the mammalian retina

Rod photoreceptors were recently shown to contact Off cone bipolar cells, providing a novel pathway for rod signal flow in the mammalian retina. By recording from pairs of rods and Off cone bipolar cells in the ground squirrel, we measured the synaptic responses of mammalian rods unfiltered by the slow kinetics of the rod bipolar cell response. We show that vesicle fusion and turnover in mammalian rods is fast, and that this new pathway can mediate rapid signaling.

Genetic Dissection of Rod and Cone Pathways in the Dark-Adapted Mouse Retina

A monumental task of the mammalian retina is to encode an enormous range (>10(9)-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod-bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [WT, Cx36(-/-), Bhlhb4(-/-), and Tralpha(-/-)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod-cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina.

Dark adaptation recovery of human rod bipolar cell response kinetics estimated from scotopic b‐wave measurements

We recorded ganzfeld scotopic ERGs to examine the responses of human rod bipolar cells in vivo, during dark adaptation recovery following bleaching exposures, as well as during adaptation to steady background lights. In order to be able to record responses at relatively early times in recovery, we utilized a 'criterion response amplitude' protocol in which the test flash strength was adjusted to elicit responses of nearly constant amplitude. In order to provide accurate and unbiased measures of response kinetics, we utilized a curve-fitting procedure to fit a smooth function to the measured responses in the vicinity of the peak, thereby extracting both the time-to-peak and the amplitude of the responses. Following bleaching exposures, the responses exhibited both desensitization and accelerated kinetics. During early post-bleach recovery, the flash sensitivity and time-to-peak varied according to a power-law expression (with an exponent of 6), as found in the presence of steady background light. This light-like phenomenon, however, appeared to be set against the backdrop of a second, more slowly recovering 'pure' desensitization, most clearly evident at late post-bleach times. The post-bleach 'equivalent background intensity' derived from measurements of flash sensitivity faded initially with an S2 slope of approximately 0.24 decades min(-1), and later as a gentle S3 tail. When calculated from kinetics, the results displayed only the S2 slope. While the recovery of rod bipolar cell response kinetics can be described accurately by a declining level of opsin in the rods, the sensitivity of these cells is reduced further than expected by this mechanism alone.

Rod and cone contributions to the a-wave of the electroretinogram of the macaque.

The electroretinogram (ERG) of anaesthetised dark-adapted macaque monkeys was recorded in response to ganzfeld stimulation and rod- and cone-driven receptoral and postreceptoral components were separated and modelled. The test stimuli were brief (< 4.1 ms) flashes. The cone-driven component was isolated by delivering the stimulus shortly after a rod-saturating background had been extinguished. The rod-driven component was derived by subtracting the cone-driven component from the mixed rod-cone ERG. The initial part of the leading edge of the rod-driven a-wave scaled linearly with stimulus energy when energy was sufficiently low and, for times less than about 12 ms after the stimulus, it was well described by a linear model incorporating a distributed delay and three cascaded low-pass filter elements. Addition of a simple static saturating non-linearity with a characteristic intermediate between a hyperbolic and an exponential function was sufficient to extend application of the model to most of the leading edge of the saturated responses to high energy stimuli. It was not necessary to assume involvement of any other non-linearity or that any significant low-pass filter followed the non-linear stage of the model. A negative inner-retinal component contributed to the later part of the rod-driven a-wave. After suppressing this component by blocking ionotropic glutamate receptors, the entire a-wave up to the time of the first zero-crossing scaled with stimulus energy and was well described by summing the response of the rod model with that of a model describing the leading edge of the rod-bipolar cell response. The negative inner-retinal component essentially cancelled the early part of the rod-bipolar cell component and, for stimuli of moderate energy, made it appear that the photoreceptor current was the only significant component of the leading edge of the a-wave. The leading edge of the cone-driven a-wave included a slow phase that continued up to the peak, and was reduced in amplitude either by a rod-suppressing background or by the glutamate analogue, cis-piperidine-2,3-dicarboxylic acid (PDA). Thus the slow phase represents a postreceptoral component present in addition to a fast component of the a-wave generated by the cones themselves. At high stimulus energies, it appeared less than 5 ms after the stimulus. The leading edge of the cone-driven a-wave was adequately modelled as the sum of the output of a cone photoreceptor model similar to that for rods and a postreceptoral signal obtained by a single integration of the cone output. In addition, the output of the static non-linear stage in the cone model was subject to a low-pass filter with a time constant of no more than 1 ms. In conclusion, postreceptoral components must be taken into account when interpreting the leading edge of the rod- and cone-driven a-waves of the dark-adapted ERG.

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.

Light-evoked responses of bipolar cells in a mammalian retina.

We recorded light-evoked responses from rod and cone bipolar cells using patch-clamp techniques in a slice preparation of the rat retina. Rod bipolar cells responded to light with a sustained depolarization (ON response) followed at light offset by a slight hyperpolarization. ON and OFF cone bipolar cells were encountered, both with diverse temporal properties. The responses of rod bipolar cells were composed primarily of two components, a nonspecific cation current and a chloride current. The chloride current was reduced greatly in axotomized cells and could be suppressed by coapplication of the GABA(A) antagonist bicuculline and the GABA(C) antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. This suggests that it largely reflects feedback from GABAergic amacrine cells. The response latency of intact rod bipolar cells was shorter than that of the axotomized cells, and the sensitivity curve covered more than twice the dynamic range. Application of the GABA receptor antagonists partially mimicked the effects of axotomy. These findings suggest that functional properties of the axon terminal system-notably synaptic feedback from amacrine cells-play an important role in defining the response properties of mammalian bipolar cells.

Responses of rod bipolar cells isolated from dogfish retinal slices to concentration-jumps of glutamate.

Rod on-bipolar cell light responses are mediated by a class of metabotropic glutamate receptor which is coupled via a G-protein to the control of a cGMP cascade, with cGMP acting to open cation channels, whilst off-bipolar cells possess ionotropic glutamate receptors. Whole-cell voltage-clamp recordings were obtained from on- and off-bipolar cells of dark-adapted dogfish retinal slices, identified by their light responses. Isolated cells were exposed to concentration-jumps of glutamate. At negative voltage-clamp potentials, on-bipolar cells responded to glutamate with outward currents with a mean delay of 10.8 ms, whilst off-bipolar cells responded with inward currents without any delay. Neither cell type showed desensitization to applied steps of glutamate. The dose-response relation for on-bipolar cells showed no gradual saturation, but increased linearly with a sharp cutoff above 200 microM glutamate. This dose-response relation could be fitted with a theoretical expression assuming Michaelis-Menten kinetics for the action of glutamate on receptors and a linear relation between the concentration of receptors bound to glutamate and the fall in cGMP this induces. The dose-response relation of off-bipolar cells showed saturation with a limiting slope of 2 at low glutamate concentrations, suggesting that two molecules of glutamate are required to open each channel by a cooperative mechanism. The glutamate receptor coupled cGMP cascade of rod on-bipolar cells can account for high synaptic voltage gain.

Responses of rod bipolar cells isolated from the rat retina to the glutamate agonist 2-amino-4-phosphonobutyric acid (APB)

Isolated rod bipolar cells were obtained by enzymatic (papain) and mechanical dissociation of the adult rat retina. Virtually all intact bipolar cells in the dissociates expressed protein kinase C (PKC) immunoreactivity, a selective marker for rod bipolar cells in the in vivo retina. Whole-cell recordings were performed using nystatin in the patch pipette to minimize washout of those cytoplasmic components necessary for the maintenance of ionic currents. At holding potentials of -33 mV, a tonic inward current was observed. The glutamate agonist 2-amino-4-phosphonobutyrate (APB) reduced this current by closing ion channels. Under normal conditions, Na+ appeared to be the main charge carrier. Both the internal and the external Ca2+ concentrations were found to exert a powerful influence on the APB-sensitive current. We conclude that the rod bipolar cell in situ is depolarized at light onset.