Arginyltransferase (Ate1) regulates the RGS7 protein level and the sensitivity of light-evoked ON-bipolar responses.

Regulator of G-protein signaling 7 (RGS7) is predominately present in the nervous system and is essential for neuronal signaling involving G-proteins. Prior studies in cultured cells showed that RGS7 is regulated via proteasomal degradation, however no protein is known to facilitate proteasomal degradation of RGS7 and it has not been shown whether this regulation affects G-protein signaling in neurons. Here we used a knockout mouse model with conditional deletion of arginyltransferase (Ate1) in the nervous system and found that in retinal ON bipolar cells, where RGS7 modulates a G-protein to signal light increments, deletion of Ate1 raised the level of RGS7. Electroretinographs revealed that lack of Ate1 leads to increased light-evoked response sensitivities of ON-bipolar cells, as well as their downstream neurons. In cultured mouse embryonic fibroblasts (MEF), RGS7 was rapidly degraded via proteasome pathway and this degradation was abolished in Ate1 knockout MEF. Our results indicate that Ate1 regulates RGS7 protein level by facilitating proteasomal degradation of RGS7 and thus affects G-protein signaling in neurons.

Range, routing and kinetics of rod signaling in primate retina.

Stimulus- or context-dependent routing of neural signals through parallel pathways can permit flexible processing of diverse inputs. For example, work in mouse shows that rod photoreceptor signals are routed through several retinal pathways, each specialized for different light levels. This light-level-dependent routing of rod signals has been invoked to explain several human perceptual results, but it has not been tested in primate retina. Here, we show, surprisingly, that rod signals traverse the primate retina almost exclusively through a single pathway - the dedicated rod bipolar pathway. Identical experiments in mouse and primate reveal substantial differences in how rod signals traverse the retina. These results require reevaluating human perceptual results in terms of flexible computation within this single pathway. This includes a prominent speeding of rod signals with light level - which we show is inherited directly from the rod photoreceptors themselves rather than from different pathways with distinct kinetics.

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full-field light of various intensities. Light-evoked membrane potential changes (ΔVm ) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark-adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage-gated Ca2+ influx (QCa ) and consequent glutamate release. Stimulus-evoked glutamate release was quantified by the increase in membrane capacitance (ΔCm ). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔVm /QCa ratio equally at a given light intensity and inhibition did not alter the overall relation between QCa and ΔCm . However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔCm unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non-linear relationship between QCa and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.

Homeostatic plasticity shapes the visual system's first synapse.

Vision in dim light depends on synapses between rods and rod bipolar cells (RBCs). Here, we find that these synapses exist in multiple configurations, in which single release sites of rods are apposed by one to three postsynaptic densities (PSDs). Single RBCs often form multiple PSDs with one rod; and neighboring RBCs share ~13% of their inputs. Rod-RBC synapses develop while ~7% of RBCs undergo programmed cell death (PCD). Although PCD is common throughout the nervous system, its influences on circuit development and function are not well understood. We generate mice in which ~53 and ~93% of RBCs, respectively, are removed during development. In these mice, dendrites of the remaining RBCs expand in graded fashion independent of light-evoked input. As RBC dendrites expand, they form fewer multi-PSD contacts with rods. Electrophysiological recordings indicate that this homeostatic co-regulation of neurite and synapse development preserves retinal function in dim light.

Dopamine Regulation of GABAA Receptors Contributes to Light/Dark Modulation of the ON-Cone Bipolar Cell Receptive Field Surround in the Retina.

Cone bipolar cells are interneurons that receive synaptic input from cone photoreceptor cells and provide the output of the first synaptic layer of the retina. These cells exhibit center-surround receptive fields, a prototype of lateral inhibition between neighboring sensory cells in which stimulation of the receptive field center excites the cell whereas stimulation of the surrounding region laterally inhibits the cell. This fundamental sensory coding mechanism facilitates spatial discrimination and detection of stimulus edges. However, although it is well established that the receptive field surround is strongest when ambient or background illumination is most intense, e.g., at midday, and that the surround is minimal following maintained darkness, the synaptic mechanisms that produce and modulate the surround have not been resolved. Using electrical recording of bipolar cells under experimental conditions in which the cells exhibited surround light responses, and light and electron microscopic immunocytochemistry, we show in the rabbit retina that bright-light-induced activation of dopamine D1 receptors located on ON-center cone bipolar cell dendrites increases the expression and activity of GABAA receptors on the dendrites of the cells and that surround light responses depend on endogenous GABAA receptor activation. We also show that maintained darkness and D1 receptor blockade following maintained illumination and D1 receptor activation result in minimal GABAA receptor expression and activity and greatly diminished surrounds. Modulation of the D1/GABAA receptor signaling pathway of ON-cBC dendrites by the ambient light level facilitates detection of spatial details on bright days and large dim objects on moonless nights.

Inhibition decorrelates visual feature representations in the inner retina.

The retina extracts visual features for transmission to the brain. Different types of bipolar cell split the photoreceptor input into parallel channels and provide the excitatory drive for downstream visual circuits. Mouse bipolar cell types have been described at great anatomical and genetic detail, but a similarly deep understanding of their functional diversity is lacking. Here, by imaging light-driven glutamate release from more than 13,000 bipolar cell axon terminals in the intact retina, we show that bipolar cell functional diversity is generated by the interplay of dendritic excitatory inputs and axonal inhibitory inputs. The resulting centre and surround components of bipolar cell receptive fields interact to decorrelate bipolar cell output in the spatial and temporal domains. Our findings highlight the importance of inhibitory circuits in generating functionally diverse excitatory pathways and suggest that decorrelation of parallel visual pathways begins as early as the second synapse of the mouse visual system.

The TRPM1 channel in ON-bipolar cells is gated by both the α and the ßγ subunits of the G-protein Go.

Transmission from photoreceptors to ON bipolar cells in mammalian retina is mediated by a sign-inverting cascade. Upon binding glutamate, the metabotropic glutamate receptor mGluR6 activates the heterotrimeric G-protein Gαoß3γ13, and this leads to closure of the TRPM1 channel (melastatin). TRPM1 is thought to be constitutively open, but the mechanism that leads to its closure is unclear. We investigated this question in mouse rod bipolar cells by dialyzing reagents that modify the activity of either Gαo or Gßγ and then observing their effects on the basal holding current. After opening the TRPM1 channels with light, a constitutively active mutant of Gαo closed the channel, but wild-type Gαo did not. After closing the channels by dark adaptation, phosducin or inactive Gαo (both sequester Gßγ) opened the channel while the active mutant of Gαo did not. Co-immunoprecipitation showed that TRPM1 interacts with Gß3 and with the active and inactive forms of Gαo. Furthermore, bioluminescent energy transfer assays indicated that while Gαo interacts with both the N- and the C- termini of TRPM1, Gßγ interacts only with the N-terminus. Our physiological and biochemical results suggest that both Gαo and Gßγ bind TRPM1 channels and cooperate to close them.

Possible roles of glutamate transporter EAAT5 in mouse cone depolarizing bipolar cell light responses.

A remarkable feature of neuronal glutamate transporters (EAATs) is their dual functions of classical carriers and ligand-gated chloride (Cl(-)) channels. Cl(-) conductance is rapidly activated by glutamate in subtype EAAT5, which mediates light responses in depolarizing bipolar cells (DBC) in retinae of lower vertebrates. In this study, we examine whether EAAT5 also mediates the DBC light response in mouse. We took advantage of an infrared illuminated micro-injection system, and studied the effects of the EAAT blocker (TBOA) and a glutamate receptor agonist (LAP4) on the mouse electroretinogram (ERG) b-wave responses. Our results showed that TBOA and LAP4 shared similar temporal patterns of inhibition: both inhibited the ERG b-wave shortly after injection and recovered with similar time courses. TBOA inhibited the b-wave completely at mesopic light intensity with an IC50 value about 1 log unit higher than that of LAP4. The inhibitory effects of TBOA and LAP4 were found to be additive in the photopic range. Furthermore, TBOA alone inhibited the b-wave in the cone operative range in knockout mice lacking DBCRs at a low concentration that did not alter synaptic glutamate clearance activity. It also produced a stronger inhibition than that of LAP4 on the cone-driven b-wave measured with a double flash method in wildtype mice. These electrophysiological data suggest a significant role for EAAT5 in mediating cone-driven DBC light responses. Our immunohistochemistry data indicated the presence of postsynaptic EAAT5 on some DBCCs and some DBCRs, providing an anatomical basis for EAAT5's role in DBC light responses.

An allosteric regulator of R7-RGS proteins influences light-evoked activity and glutamatergic waves in the inner retina.

In the outer retina, G protein-coupled receptor (GPCR) signaling mediates phototransduction and synaptic transmission between photoreceptors and ON bipolar cells. In contrast, the functions of modulatory GPCR signaling networks in the inner retina are less well understood. We addressed this question by determining the consequences of augmenting modulatory Gi/o signaling driven by endogenous transmitters. This was done by analyzing the effects of genetically ablating the R7 RGS-binding protein (R7BP), a membrane-targeting protein and positive allosteric modulator of R7-RGS (regulator of the G protein signaling 7) family that deactivates Gi/oα subunits. We found that R7BP is expressed highly in starburst amacrine cells and retinal ganglion cells (RGCs). As indicated by electroretinography and multielectrode array recordings of adult retina, ablation of R7BP preserved outer retina function, but altered the firing rate and latency of ON RGCs driven by rods and cones but not rods alone. In developing retina, R7BP ablation increased the burst duration of glutamatergic waves whereas cholinergic waves were unaffected. This effect on glutamatergic waves did not result in impaired segregation of RGC projections to eye-specific domains of the dorsal lateral geniculate nucleus. R7BP knockout mice exhibited normal spatial contrast sensitivity and visual acuity as assessed by optomotor reflexes. Taken together these findings indicate that R7BP-dependent regulation of R7-RGS proteins shapes specific aspects of light-evoked and spontaneous activity of RGCs in mature and developing retina.

A time and cost efficient approach to functional and structural assessment of living neuronal tissue.

In this manuscript, we describe a protocol for capturing both physiological and structural properties of living neuronal tissue. An essential aspect of this method is its flexibility and fast turnaround time. It is a streamlined process that includes recording of electrophysiological neuronal activity, calcium imaging, and structural analysis. This is accomplished by placing intact tissue on a modified Millicell Biopore insert. The Biopore membrane suspends the tissue in the perfusion solution, allowing for complete access to nutrients, oxygen, and pharmacological agents. The ring that holds the membrane ensures its structural stability; forceps can be used to grip the ring without contacting the filter or the tissue, for easy transfer between multiple setups. We show that tissue readily adheres to the surface of the membrane, its entire surface is visible in transmitted light and accessible for recording and imaging, and remains responsive to physiological stimuli for extended periods of time.

Rod and cone pathway signalling is altered in the P2X7 receptor knock out mouse.

The P2X7 receptor (P2X7-R) is expressed in the retina and brain and has been implicated in neurodegenerative diseases. However, whether it is expressed by neurons and plays a role as a neurotransmitter receptor has been the subject of controversy. In this study, we first show that the novel vesicular transporter for ATP, VNUT, is expressed in the retina, verifying the presence of the molecular machinery for ATP to act as neurotransmitter at P2X7-Rs. Secondly we show the presence of P2X7-R mRNA and protein in the retina and cortex and absence of the full length variant 1 of the receptor in the P2X7-R knock out (P2X7-KO) mouse. The role of the P2X7-R in neuronal function of the retina was assessed by comparing the electroretinogram response of P2X7-KO with WT mice. The rod photoreceptor response was found to be similar, while both rod and cone pathway post-photoreceptor responses were significantly larger in P2X7-KO mice. This suggests that activation of P2X7-Rs modulates output of second order retinal neurons. In line with this finding, P2X7-Rs were found in the outer plexiform layer and on inner retinal cell classes, including horizontal, amacrine and ganglion cells. The receptor co-localized with conventional synapses in the IPL and was expressed on amacrine cells post-synaptic to rod bipolar ribbon synapses. In view of the changes in visual function in the P2X7-KO mouse and the immunocytochemical location of the receptor in the normal retina, it is likely the P2X7-R provides excitatory input to photoreceptor terminals or to inhibitory cells that shape both the rod and cone pathway response.

A mammalian retinal bipolar cell uses both graded changes in membrane voltage and all-or-nothing Na+ spikes to encode light.

Barlow (1953) studied summation in ganglion cell receptive fields and observed a fine discrimination of spatial information from which he inferred that retinal interneurons use analog signals to process images. Subsequent intracellular recordings confirmed that the interneurons of the outer retina, including photoreceptors, horizontal cells, and bipolar cells, respond to light with slow, graded changes in membrane potential. Analog processing may enable interneurons to discriminate fine gradations in light intensity and spatiotemporal pattern, but at the expense of the speed, temporal precision, and threshold discrimination that are characteristic of all-or-nothing Na(+) spikes. We show that one type of mammalian On bipolar cell, the ground squirrel cb5b, has a large tetrodotoxin (TTX)-sensitive Na(+) current. When recorded from in the perforated patch configuration, cb5b cells can signal the onset of a light step with 1-3 all-or-nothing action potentials that attain a peak amplitude of -10 to -20 mV (peak width at half-height equals 2-3 ms). When exposed to a continuous, temporally fluctuating stimulus, cb5b cells generate both graded and spiking responses. Cb5b cells spike with millisecond precision, selecting for stimulus sequences in which transitions to light are preceded by a period of darkness. The axon terminals of cb5b bipolar cells costratify with the dendrites of amacrine and ganglion cells that encode light onset with a short latency burst of spikes. The results support the idea that a spiking On bipolar cell is part of a dedicated retinal pathway for rapidly and reliably signaling dark to light transitions.

RGS9 knockout causes a short delay in light responses of ON-bipolar cells.

RGS9 and R9AP are components of the photoreceptor-specific GTPase activating complex responsible for rapid inactivation of the G protein, transducin, in the course of photoresponse recovery from excitation. The amount of this complex in photoreceptors is strictly dependent on the expression level of R9AP; consequently, the knockouts of either RGS9 or R9AP cause comparable delays in photoresponse recovery. While RGS9 is believed to be present only in rods and cones, R9AP is also expressed in dendritic tips of ON-bipolar cells, which receive synaptic inputs from photoreceptors. Recent studies demonstrated that knockouts of R9AP and its binding partner in ON-bipolar cells, RGS11, cause a small delay in ON-bipolar cell light responses manifested as a delayed onset of electroretinography b-waves. This led the authors to suggest that R9AP and RGS11 participate in regulating the kinetics of light responses in these cells. Here we report the surprising finding that a nearly identical b-wave delay is observed in RGS9 knockout mice. Given the exclusive localization of RGS9 in photoreceptors, this result argues for a presynaptic origin of the b-wave delay in this case and perhaps in the case of the R9AP knockout as well, since R9AP is expressed in both photoreceptors and ON-bipolar cells. We also conducted a detailed analysis of the b-wave rising phase kinetics in both knockout animal types and found that, despite a delayed b-wave onset, the slope of the light response is unaffected or increased, dependent on the light stimulus intensity. This result is inconsistent with a slowdown of response propagation in ON-bipolar cells caused by the R9AP knockout, further arguing against the postsynaptic nature of the delayed b-wave phenotype in RGS9 and R9AP knockout mice.

Minor effect of blue-light filtering on multifocal electroretinograms.

PURPOSE: To assess the impact of blue-light filtering on retinal processing to evaluate potential side effects of these filters on visual function at the neural level. SETTING: Department of Ophthalmology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany. DESIGN: Cohort study. METHODS: Multifocal electroretinograms (ERGs) were recorded monocularly after pupil dilation in pseudophakic patients with a colorless intraocular lens (IOL) under 2 conditions: (1) stimulus perception through a yellow-tinted filter with the filter characteristics of the AF-1 YA-60BB IOL (blue-light filter) and (2) stimulus perception through a neutral filter that homogeneously attenuates the effective stimulus intensity like the blue-light filter independent of the wavelength. First-order kernel multifocal ERGs were extracted at 61 visual field locations and averaged for 5 stimulus eccentricities. Amplitudes and implicit times were determined for the multifocal ERG components N1 (first negative deflection), N2 (second negative deflection), and P1 (first positive deflection). RESULTS: The study evaluated 20 patients. Typical multifocal ERGs were obtained for both conditions at all eccentricities. There were no significant differences in amplitudes or implicit times between the 2 conditions except for a slight P1 amplitude enhancement (6.9%) with the blue-light filter at an intermediate eccentricity (P = .003). CONCLUSIONS: The bipolar cell-dominated multifocal ERG was largely unaffected by short-term effects of blue-light filtering. The induced change in the spectral composition of the stimulus did not significantly alter the activity at the input stage of the visual system, specifically the retinal network comprising photoreceptors, horizontal cells, and bipolar cells.

PKC{alpha} is essential for the proper activation and termination of rod bipolar cell response.

PURPOSE: Protein kinase (PKC)-α is abundant in retinal bipolar cells. This study was performed to explore its role in visual processing. METHODS: PKCα-knockout (Prkca(-/-)) mice and control animals were examined by using electroretinography (ERG), light microscopy, and immunocytochemistry. RESULTS: 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. CONCLUSIONS: 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.

Phosducin regulates transmission at the photoreceptor-to-ON-bipolar cell synapse.

The rate of synaptic transmission between photoreceptors and bipolar cells has been long known to depend on conditions of ambient illumination. However, the molecular mechanisms that mediate and regulate transmission at this ribbon synapse are poorly understood. We conducted electroretinographic recordings from dark- and light-adapted mice lacking the abundant photoreceptor-specific protein phosducin and found that the ON-bipolar cell responses in these animals have a reduced light sensitivity in the dark-adapted state. Additional desensitization of their responses, normally caused by steady background illumination, was also diminished compared with wild-type animals. This effect was observed in both rod- and cone-driven pathways, with the latter affected to a larger degree. The underlying mechanism is likely to be photoreceptor specific because phosducin is not expressed in other retina neurons and transgenic expression of phosducin in rods of phosducin knock-out mice rescued the rod-specific phenotype. The underlying mechanism functions downstream from the phototransduction cascade, as evident from the sensitivity of phototransduction in phosducin knock-out rods being affected to a much lesser degree than b-wave responses. These data indicate that a major regulatory component responsible for setting the sensitivity of signal transmission between photoreceptors and ON-bipolar cells is confined to photoreceptors and that phosducin participates in the underlying molecular mechanism.

RGS7 and -11 complexes accelerate the ON-bipolar cell light response.

PURPOSE: The retinal ON-bipolar cell (ON-BPC) light response is initiated upon deactivation of the metabotropic glutamate receptor mGluR6 and the G protein Go. G protein-based signaling cascades are typically accelerated by interaction of the G protein alpha subunit with a member of the regulator of G protein signaling (RGS) protein family. The goal of this study was to determine whether RGS7 and/or -11 serve this function in retinal ON-BPCs. METHODS: Retinas from mice lacking RGS11 (RGS11(-/-)), or with a deletion mutation in RGS7 (RGS7(Delta/Delta)), or both, were compared to wild-type (WT) by immunofluorescence confocal microscopy. The retinal light response was measured with the electroretinogram (ERG). The kinetics of simulated light responses from individual rod bipolar cells were recorded by whole-cell patch-clamp electrophysiology. RESULTS: Levels of the R7 RGS interaction partners, Gbeta5 and R9AP, were reduced in the outer plexiform layer of the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. ERG recordings demonstrated a delay in the rising phase of the ERG b-wave, larger photopic b-wave amplitudes, and increased scotopic threshold response sensitivity in the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. The ERG measured from the RGS7(Delta/Delta) retina was normal. Patch-clamp recordings of chemically simulated light responses of rod BPCs revealed a 25-ms delay in the onset of the ON-BPC response in the RGS7(Delta/Delta)/RGS11(-/-) mouse compared with the WT. CONCLUSIONS: RGS11 plays a role in the deactivation of Galphao, which precedes activation of the depolarizing current in ON-BPCs. RGS7 must also serve a role as changes in RGS7(Delta/Delta)/RGS11(-/-) mice were greater than those in RGS11(-/-) mice.

A spectral model for signal elements isolated from zebrafish photopic electroretinogram.

The zebrafish photopic electroretinogram (ERG) sums isolatable elements. In each element, red-, blue-, green-, and UV- (r, g, b, and u) cone signals combine in a way that reflects retinal organization. ERG responses to monochromatic stimuli of different wavelengths and irradiances were recorded on a white rod suppressing background using superfused eyecups. Onset elements were isolated with glutamatergic blockers and response subtractions. CNQX-blocked ionotropic (AMPA/kainate) glutamate receptors; l-AP4 or CPPG-blocked metabotropic (mGluR6) glutamate receptors; TBOA-blocked glutamate transporters; and l-aspartate inactivated all glutamatergic mechanisms. Seven elements emerged: photopic PIII, the l-aspartate-isolated cone response; b1, a CNQX-sensitive early b-wave element of inner retinal origin; PII, a photopic, CNQX-insensitive composite b-wave element from ON bipolar cells; PIIm, an l-AP4/CPPG-sensitive, CNQX-insensitive, metabotropic subelement of PII; PIInm, an l-AP4/CPPG/CNQX-insensitive nonmetabotropic subelement of PII; a1nm, a TBOA-sensitive, CNQX/l-AP4/CPPG-insensitive, nonmetabotropic, postphotoreceptor a-wave element; and a2, a CNQX-sensitive a-wave element linked to OFF bipolar cells. The first five elements were fit with a spectral model that demonstrates independence of cone-color pathways. From this, Vmax and half-saturation values (k) for the contributing r-, g-, b-, and u-cone signals were calculated. Two signal patterns emerged. For PIII or PIInm, the Vmax order was Vr > Vg >> Vb approximately Vu. For b1, PII, and PIIm, the Vmax order was Vr approximately Vb > Vg > Vu. In either pattern, u-cone amplitude (Vu) was smallest, but u-cone sensitivity (ku362) was greatest, some 10-30 times greater than r cone (kr570). The spectra of b1/PII/PIIm elements peaked near b- and u-cone absorbance maxima regardless of criteria, but the spectra of PIII/PIInm elements shifted from b- toward r-cone absorbance maxima as criterion levels increased. The greatest gains in Vmax relative to PIII occurred for the b- and u-cone signals in the b1/PII/PIIm b-wave elements. This suggests a high-gain prolific metabotropic circuitry for b- and u-cone bipolar cells.

Light responses in the mouse retina are prolonged upon targeted deletion of the HCN1 channel gene.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels contribute to pacemaker activity, and co-determine the integrative behaviour of neurons and shape their response to synaptic stimulation. Four channel isoforms, HCN1-4, have been described in mammals. Recent studies showed particularly strong expression of HCN1 channels in rods and cones of the rat retina, suggesting that HCN1 channels are involved in the shaping of light responses in both types of photoreceptors. Therefore, the loss of HCN1 channels should lead to pronounced changes in light-induced electrical responses under both scotopic and photopic conditions. This was tested using a mouse transgenic approach. We used immunohistochemistry and patch-clamp recording to study the distribution of HCN1 channels in the mouse retina. HCN1 channels were strongly expressed in rod and cone photoreceptors, as well as in some bipolar, amacrine and ganglion cell types. In electroretinograms (ERGs) from animals in which the HCN1 channel gene had been knocked out, the b-wave amplitudes were unaltered (scotopic conditions) or somewhat reduced (photopic conditions), whereas the duration of both scotopic and photopic ERG responses was strikingly prolonged. Our data suggest that in visual information processing, shortening and shaping of light responses by activation of HCN1 at the level of the photoreceptors is an important step in both scotopic and photopic pathways.

Retinal ON bipolar cells express a new PCP2 splice variant that accelerates the light response.

PCP2, a member of the GoLoco domain-containing family, is present exclusively in cerebellar Purkinje cells and retinal ON bipolar cells. Its function in these tissues is unknown. Biochemical and expression system studies suggest that PCP2 is a guanine nucleotide dissociation inhibitor, although a guanine nucleotide exchange factor has also been suggested. Here, we studied the function of PCP2 in ON bipolar cells because their light response depends on Galpha(o1), which is known to interact with PCP2. We identified a new splice variant of PCP2 (Ret-PCP2) and localized it to rod bipolar and ON cone bipolar cells. Electroretinogram recordings from PCP2-null mice showed a normal a-wave but a slower falling phase of the b-wave (generated by the activity of ON bipolar cells) relative to the wild type. Whole-cell recordings from rod bipolar cells showed, both under Ames medium and after blocking GABA(A/C) and glycine receptors, that PCP2-null rod bipolar cells were more depolarized than wild-type cells with greater inward current when clamped to -60 mV. Also under both conditions, the rise time of the response to intense light was slower by 28% (Ames) and 44% (inhibitory blockers) in the null cells. Under Ames medium, we also observed >30% longer decay time in the PCP2-null rod bipolar cells. We conclude that PCP2 facilitates cation channels closure in the dark, shortens the rise time of the light response directly, and accelerates the decay time indirectly via the inhibitory network. These data can most easily be explained if PCP2 serves as a guanine nucleotide exchange factor.