Nitric oxide (NO) is a neuromodulator involved in physiological and pathological processes in the retina. In the inner retina, a subgroup of amacrine cells have been shown to synthesize NO, but bipolar cells remain controversial as NO sources. This study correlates NO synthesis in dark-adapted retinas, through labeling with the NO marker DAF-FM, with neuronal nitric oxide synthase (nNOS) and inducible NOS expression, and presence of the NO receptor soluble guanylate cyclase in bipolar cells. NO containing bipolar cells were morphologically identified by dialysis of DAF fluorescent cells with intracellular dyes, or by DAF labeling followed by immunohistochemistry for nNOS and other cellular markers. DAF fluorescence was observed in all types of bipolar cells that could be identified, but the most intense DAF fluorescence was observed in bipolar cells with severed processes, supporting pathological NO signaling. Among nNOS expressing bipolar cells, type 9 was confirmed unequivocally, while types 2, 3a, 3b, 4, 5, 7, 8 and the rod bipolar cell were devoid of this enzyme. These results establish specific bipolar cell types as NO sources in the inner retina, and support the involvement of NO signaling in physiological and pathological processes in the inner retina.

Some mammalian rod bipolar cells (RBCs) can receive excitatory chemical synaptic inputs from both rods and cones (DBCR2 ), but anatomical evidence for mammalian cone-RBC contacts has been sparse. We examined anatomical cone-RBC contacts using neurobiotin (NB) to visualize individual mouse cones and standard immuno-markers to identify RBCs, cone pedicles and synapses in mouse and baboon retinas. Peanut agglutinin (PNA) stained the basal membrane of all cone pedicles, and mouse cones were positive for red/green (R/G)-opsin, whereas baboon cones were positive for calbindin D-28k. All synapses in the outer plexiform layer were labeled for synaptic vesicle protein 2 (SV2) and PSD (postsynaptic density)-95, and those that coincided with PNA resided closest to bipolar cell somas. Cone-RBC synaptic contacts were identified by: (a) RBC dendrites deeply invaginating into the center of cone pedicles (invaginating synapses), (b) RBC dendritic spines intruding into the surface of cone pedicles (superficial synapses), and (c) PKCα immunoreactivity coinciding with synaptic marker SV2, PSD-95, mGluR6, G protein beta 5 or PNA at cone pedicles. One RBC could form 0-1 invaginating and 1-3 superficial contacts with cones. 20.7% and 38.9% of mouse RBCs contacted cones in the peripheral and central retina (p < .05, n = 14 samples), respectively, while 34.4% (peripheral) and 48.5% (central) of cones contacted RBCs (p > .05). In baboon retinas (n = 4 samples), cone-RBC contacts involved 12.2% of RBCs (n = 416 cells) and 22.5% of cones (n = 225 cells). This suggests that rod and cone signals in the ON pathway are integrated in some RBCs before reaching AII amacrine cells.

Endocytosis and exocytosis were investigated in the synaptic terminal of retinal bipolar cells by monitoring the uptake and loss of the fluorescent dye FM1-43. Depolarization in the presence of Ca2+ stimulated a continuous cycle of exocytosis and endocytosis that was approximately balanced at rates up to 3800 vesicles per s. Vesicles became available for exocytosis within 1 min of endocytosis, and about 700,000 releasable vesicles were specifically localized to a region within 2 microm of the plasma membrane. Release of caged Ca2+ using NP-EGTA while simultaneously monitoring cytosolic Ca2+ with Fura-2 indicated that continuous exocytosis was stimulated by sub-micromolar levels of Ca2+. It has been suggested that the ribbon synapse of bipolar cells only supports transient exocytosis, but our results demonstrate that this synapse is specialized for the continuous secretion of neurotransmitter.

Retinal bipolar and amacrine cells receive visual information from photoreceptors and participate in the first steps of image processing in the retina. Several studies have suggested the operation of aerobic glycolysis and a lactate shuttle system in the retina due to the high production of this metabolite under aerobic conditions. However, whether bipolar cells form part of this metabolic circuit remains unclear. Here, we show that the monocarboxylate transporter 2 is expressed and functional in inner retinal neurons. Additionally, we used genetically encoded FRET nanosensors to demonstrate the ability of inner retinal neurons to consume extracellular lactate as an alternative to glucose. In rod bipolar cells, lactate consumption allowed cells to maintain the homeostasis of ions and electrical responses. We also found that lactate synthesis and transporter inhibition caused functional alterations and an increased rate of cell death. Overall, our data shed light on a notable but still poorly understood aspect of retinal metabolism.

One general categorization of retinal ganglion cells is to segregate them into tonically or phasically responding neurons, each conveying discrete aspects of the visual scene. Although best identified in the output signals of the retina, this distinction is initiated at the first synapse: between photoreceptors and the dendrites of bipolar cells. In this study we found that the output synapses of bipolar cells also contribute to separate these pathways. Both transient and sustained ganglion cells can produce maintained spike activity, but bipolar cell glutamate release exhibits a divergence that corresponds to the response characteristics of the ganglion cells. Comparing light intensity coding in the sustained and transient ON pathways revealed that they shared the intensity spectrum. The transient pathway had greater sensitivity but smaller dynamic range, and switched from intensity coding to event detection at light levels where sustained pathway sensitivity began to rise. The distinctive properties of the sustained pathway depended upon inhibition and shifted toward those of the transient pathway in the absence of inhibition. The transient system was comparatively unaffected by the loss of inhibition and this was due to the concomitant activation of perisynaptic NMDA receptors. Overall, the properties of bipolar cell dendritic and axon terminals both contribute to the formation of key aspects of the sustained/transient dichotomy normally associated with ganglion cells.

The balance of neuronal excitation and inhibition is important for proper retinal signaling. A previous report showed that diabetes selectively reduces light-evoked inhibition to the retinal dim light rod pathway, changing this balance. Here, changes in mechanisms of retinal inhibitory synaptic transmission after 6 weeks of diabetes are investigated.

Rod bipolar cells transmit visual signals from their dendrites, where they receive input from rod photoreceptors, to their axon terminals, where they synapse onto amacrine cells. Little is known, however, about the transmission and possible transformation of these signals. We have combined axon terminal recording in retinal slices, quantitative, light-microscopic morphological reconstruction and computer modelling to obtain detailed compartmental models of rat rod bipolar cells. Passive cable properties were estimated by directly fitting the current responses of the models evoked by voltage pulses to the physiologically recorded responses. At a holding potential of -60 mV, the average best-fit parameters were 1.1 microF cm(-2) for specific membrane capacitance (C(m)), 130 Omega cm for cytoplasmic resistivity (R(i)), and 24 kOmega cm(2) for specific membrane resistance (R(m)). The passive integration of excitatory and inhibitory synaptic inputs was examined by computer modelling with physiologically realistic synaptic conductance waveforms. For both transient and steady-state synaptic inhibition, the inhibitory effect was relatively insensitive to the location of the inhibition. For transient synaptic inhibition, the time window of effective inhibition depended critically on the relative timing of inhibition and excitation. The passive signal transmission between soma and axon terminal was examined by the electrotonic transform and quantified as the frequency-dependent voltage attenuation of sinusoidal voltage waveforms. For the range of parameters explored (axon diameter and length, R(i)), the lowest cutoff frequency observed was approximately 300 Hz, suggesting that realistic scotopic visual signals will be faithfully transmitted from soma to axon terminal, with minimal passive attenuation along the axon.

Postsynaptic kainate receptors mediate excitatory synaptic transmission over a broad range of temporal frequencies. In heterologous systems, the temporal responses of kainate receptors vary when different channel-forming and auxiliary subunits are co-expressed but how this variability relates to the temporal differences at central synapses is incompletely understood. The mammalian cone photoreceptor synapse provides advantages for comparing the different temporal signalling roles of kainate receptors, as cones release glutamate over a range of temporal frequencies, and three functionally distinct Off bipolar cell types receive cone signals at synapses that contain either AMPA or kainate receptors, all with different temporal properties. A disadvantage is that the different receptor subunits are not identified. We used in situ hybridization, immunocytochemistry, and pharmacology to identify the kainate receptor and auxiliary subunits in ground squirrel (Ictidomys tridecimlineatus) cb1a/b, cb2, and cb3a/b Off bipolar cell types. As expected, the types showed distinct subunit expression patterns. Kainate receptors mediated ∼80% of the synaptic response in cb3a/b cells and were heteromers of GluK1 and GluK5. Cb3a/b cells contained message for GluK1 and GluK5, and also GluK3 and the auxiliary subunit Neto1. The synaptic responses in cb1a/b cells were mediated by GluK1-containing kainate receptors that behaved differently from the receptors expressed by cb3a/b cells. AMPA receptors mediated the entire synaptic response in cb2 cells and the remaining synaptic response in cb3a/b cells. We conclude that GluK1 is the predominant kainate receptor subunit in cb1 and cb3 Off bipolar cells. Different temporal response properties may result from selective association with GluK3, GluK5, or Neto1.

Glutamatergic retinal waves, the spontaneous patterned neural activities propagating among developing retinal ganglion cells (RGCs), instruct the activity-dependent refinement of visuotopic maps. However, its initiation and underlying mechanism remain largely elusive. Here using larval zebrafish and multiple in vivo approaches, we discover that bipolar cells (BCs) are responsible for the generation of glutamatergic retinal waves. The wave originates from BC axon terminals (ATs) and propagates laterally to nearby BCs and vertically to downstream RGCs and the optic tectum. Its initiation is triggered by the activation of and consequent glutamate release from BC ATs, and is mediated by the N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) expressed at these ATs. Intercellular asymmetry of NMDAR expression at BC ATs enables the preferential initiation of waves at the temporal retina, where BC ATs express more NMDARs. Thus, our findings indicate that glutamatergic retinal waves are initiated by BCs through a presynaptic NMDA autoreceptor-dependent process.

The retina and the olfactory bulb are the gateways to the visual and olfactory systems, respectively, similarly using neural networks to initiate sensory signal processing. Sensory receptors receive signals that are transmitted to neural networks before projecting to primary cortices. These networks filter sensory signals based on their unique features and adjust their sensitivities by gain control systems. Interestingly, dopamine modulates sensory signal transduction in both systems. In the retina, dopamine adjusts the retinal network for daylight conditions ("light adaptation"). In the olfactory system, dopamine mediates lateral inhibition between the glomeruli, resulting in odorant signal decorrelation and discrimination. While dopamine is essential for signal discrimination in the olfactory system, it is not understood whether dopamine has similar roles in visual signal processing in the retina. To elucidate dopaminergic effects on visual processing, we conducted patch-clamp recording from second-order retinal bipolar cells, which exhibit multiple types that can convey different temporal features of light. We recorded excitatory postsynaptic potentials (EPSPs) evoked by various frequencies of sinusoidal light in the absence and presence of a dopamine receptor 1 (D1R) agonist or antagonist. Application of a D1R agonist, SKF-38393, shifted the peak temporal responses toward higher frequencies in a subset of bipolar cells. In contrast, a D1R antagonist, SCH-23390, reversed the effects of SKF on these types of bipolar cells. To examine the mechanism of dopaminergic modulation, we recorded voltage-gated currents, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and low-voltage activated (LVA) Ca2+ channels. SKF modulated HCN and LVA currents, suggesting that these channels are the target of D1R signaling to modulate visual signaling in these bipolar cells. Taken together, we found that dopamine modulates the temporal tuning of a subset of retinal bipolar cells. Consequently, we determined that dopamine plays a role in visual signal processing, which is similar to its role in signal decorrelation in the olfactory bulb.

Properties of the Ca2+ -activated K+ current (I[K(Ca)]) were investigated in bipolar cells isolated from the goldfish retina. Pharmacological experiments and single channel current recordings demonstrated that I[K(Ca)] represented currents through BK channels, which were confined mostly to the presynaptic terminal. The ensemble noise analysis of I[K(Ca)], which was evoked following the activation of presynaptic Ca2+ current, revealed that the single channel conductance and open probability (P(o)) were approximately 50 pS ([K+]o = 2.6 mM, [K+]i = 140 mM) and 0.6 at 0 mV, respectively. To estimate [Ca2+]i at the cytosolic side of BK channels, activation of I[K(Ca)] was examined in Ca2+-loaded bipolar cells bathed in Co2+ solution. [Ca2+]i was monitored using furaptra fluorimetry. It was found that [Ca2+]i ranged between 10 and 20 microM when P(o) was 0.6. A high concentration of BAPTA ( > 20 mM) was required to suppress I[K(Ca)]. Under this condition, channel number was reduced without changing P(o). Therefore, it is likely that some BK channels are co-localized with Ca2+ channels in presynaptic terminals of retinal bipolar cells.

The time course of G-protein-coupled responses is largely determined by the kinetics of GTP hydrolysis by the G protein alpha subunit, which is accelerated by interaction with regulator of G-protein signaling (RGS) proteins. Light responses of ON-bipolar cells of the vertebrate retina require rapid inactivation of the G protein Galphao, which is activated in the dark by metabotropic glutamate receptor, mGluR6, in their dendritic tips. It is not yet known, however, which RGS protein(s) might be responsible for rapid inactivation kinetics. By immunofluorescence and co-immunoprecipitation, we have identified complexes of the Galphao-selective RGS proteins RGS7 and RGS11, with their obligate binding partner, Gbeta5, that are localized to the dendritic tips of murine rod and cone ON-bipolar cells, along with mGluR6. Experiments using pre- and post-synaptic markers, and a dissociated bipolar cell preparation, clearly identified the location of these complexes as the ON-bipolar cell dendritic tips and not the adjacent photoreceptor terminals or horizontal cell dendrites. In mice lacking mGluR6, the distribution of RGS11, RGS7 and Gbeta5 shifts away from the dendritic tips, implying a functional relationship with mGluR6. The precise co-localization of Gbeta5-RGS7 and Gbeta5-RGS11 with mGluR6, and the dependence of localization on the presence of mGluR6, suggests that Gbeta5-RGS7 and Gbeta5-RGS11 function specifically in the mGluR6 signal transduction pathway, where they may stimulate the GTPase activity of Galphao, thus accelerating the ON-bipolar cell light response, in a manner analogous to the acceleration of photoreceptor light responses by the Gbeta5-RGS9-1 complex.

Retinal implants allowing blind people suffering from diseases like retinitis pigmentosa and macular degeneration to regain rudimentary vision are struggling with several obstacles. One of the main problems during external electric stimulation is the co-activation of the ON and OFF pathways which results in mutual impairment. In this study the response of ON and OFF cone retinal bipolar cells during extracellular electric stimulation from the subretinal space was examined. To gain deeper insight into the behavior of these cells sustained L-type and transient T-type calcium channels were integrated in the synaptic terminals of reconstructed 3D morphologies of ON and OFF cone bipolar cells. Intracellular calcium concentration in the synaptic regions of the model neurons was investigated as well since calcium influx is a crucial parameter for cell-to-cell activity between bipolar cells and retinal ganglion cells. It was shown that monophasic stimulation results in significant different calcium concentrations in the synaptic terminals of ON and OFF bipolar cells. Intracellular calcium increased to values up to fourfold higher in the OFF bipolar model neuron in comparison to the ON bipolar cell. Furthermore, geometric properties strongly influence the activation of bipolar cells. Monophasic, biphasic, single and repetitive pulses with similar lengths, amplitudes and polarities were applied to the two model neurons.

Retinal bipolar cells (BCs) connect with photoreceptors and relay visual information to retinal ganglion cells (RGCs). Retina-specific deletion of Lhx4 in mice results in a visual defect resembling human congenital stationary night blindness. This visual dysfunction results from the absence of rod bipolar cells (RBCs) and the loss of selective rod-connecting cone bipolar cell (CBC) subtypes and AII amacrine cells (ACs). Inactivation of Lhx4 causes the apoptosis of BCs and cell fate switch from some BCs to ACs, whereas Lhx4 overexpression promotes BC genesis. Moreover, Lhx4 positively regulates Lhx3 expression to drive the fate choice of type 2 BCs over the GABAergic ACs. Lhx4 inactivation ablates Bhlhe23 expression, whereas overexpression of Bhlhe23 partially rescues RBC development in the absence of Lhx4. Thus, by acting upstream of Bhlhe23, Prdm8, Fezf2, Lhx3, and other BC genes, Lhx4, together with Isl1, could play essential roles in regulating the subtype-specific development of RBCs and CBCs.

The chemical signal of light onset, a decrease in glutamate release from rod and cone photoreceptors, is processed by a postsynaptic G protein signaling cascade in ON-bipolar cells (BPCs). The metabotropic glutamate receptor mGluR6, along with other cascade elements, is localized synaptically at the BPC dendritic tips. The effector ion channel protein transient receptor potential melastatin-1 (TRPM1), in contrast, is located not only at the dendritic tips but also in BPC bodies and axons. Little is known about the intracellular localization of TRPM1, or its trafficking route to the dendritic tip plasma membrane. Recombinant TRPM1 expressed in mammalian cells colocalized with endoplasmic reticulum (ER) markers, with little or none detected at the plasma membrane. In mouse retina, somatic TRPM1 was similarly intracellular, and not at the plasma membrane. Labeling of ER membranes by expression of a fluorescent marker showed that in BPCs the ER extends into axons and dendrites, but not dendritic tips. In cell bodies, TRPM1 colocalized with the ER, and not with the Golgi apparatus. Fluorescence protease protection (FPP) assays with TRPM1-GFP fusions in heterologous cells revealed that the N and C termini are both accessible to the cytoplasm, consistent with the transmembrane domain topology of related TRP channels. These results indicate that the majority of TRPM1 is present in the ER, from which it can potentially be transported to the dendritic tips as needed for ON light responses. The excess of ER-resident TRPM1 relative to the amount needed at the dendritic tips suggests a potential new function for TRPM1 in the ER.

The transient receptor potential vanilloid 4 (TRPV4) channel may be opened by mechanical stimuli to mediate Ca2+ and Na+ influxes, and it has been suggested to mediate glaucoma retinopathy. However, it has been mostly unclear how TRPV4 activities affect the function of primate retinal ganglion cells (RGCs). We studied RGCs and bipolar cells (BCs) in the peripheral retina of the old-world primate using whole-cell current-clamp and voltage-clamp recordings, immunomarkers and confocal microscopy. RGCs were distinguished from displaced amacrine cells (ACs) by the absence of GABA and glycine immunoreactivity and possession of an axon and a large soma in the RGC layer. Strong TRPV4 signal was concentrated in medium to large somas of RGCs, and some TRPV4 signal was found in BCs (including PKCα-positive rod BCs), as well as the end feet, soma and outer processes of Mȕller cells. TRPV4 immunoreactivity quantified by the pixel intensity histogram revealed a high-intensity component for the plexiform layers, a low-intensity component for the soma layers of ACs and Mȕller cells, and both components in the soma layers of RGCs and BCs. In large RGCs, TRPV4 agonists 4α-phorbol 12,13 didecanoate (4αPDD) and GSK1016790A reversibly enhanced the spontaneous firing and shortened the delay of voltage-gated Na+ (Nav) currents under current-clamp conditions, and under voltage-clamp conditions, 4αPDD largely reversibly increased the amplitude and frequency of spontaneous excitatory postsynaptic currents. In BCs, changes in the membrane tension induced by either applying pressure or releasing the pressure both activated a transient cation current, which reversed at ~ -10 mV and was enhanced by heating from 24 °C to 30 °C. The pressure for the half-maximal effect was ~18 mmHg. These data indicate that functional TRPV4 channels are variably expressed in primate RGCs and BCs, possibly contributing to pressure-related changes in RGCs in glaucoma.

The conversion of an analog stimulus into the digital form of spikes is a fundamental step in encoding sensory information. Here, we investigate this transformation in the visual system of fish by in vivo calcium imaging and electrophysiology of retinal bipolar cells, which have been assumed to be purely graded neurons.

Contrast is computed throughout the nervous system to encode changing inputs efficiently. The retina encodes luminance and contrast over a wide range of visual conditions and must adapt its responses to maintain sensitivity and to avoid saturation. We examined the means by which one type of adaptation allows individual synapses to compute contrast and encode luminance in biphasic responses to step changes in light levels. Light-evoked depletion of the readily releasable vesicle pool (RRP) at rod bipolar cell ribbon synapses in rat retina limited the dynamic range available to encode transient, but not sustained, responses, thereby allowing the transient and sustained components of release to compute temporal contrast and encode mean light levels, respectively. A release/replenishment model revealed that a single, homogeneous pool of synaptic vesicles is sufficient to generate this behavior and that a partial depletion of the RRP is the dominant mechanism for shaping the biphasic contrast/luminance response.

Antagonistic interactions between center and surround receptive field (RF) components lie at the heart of the computations performed in the visual system. Circularly symmetric center-surround RFs are thought to enhance responses to spatial contrasts (i.e., edges), but how visual edges affect motion processing is unclear. Here, we addressed this question in retinal bipolar cells, the first visual neuron with classic center-surround interactions. We found that bipolar glutamate release emphasizes objects that emerge in the RF; their responses to continuous motion are smaller, slower, and cannot be predicted by signals elicited by stationary stimuli. In our hands, the alteration in signal dynamics induced by novel objects was more pronounced than edge enhancement and could be explained by priming of RF surround during continuous motion. These findings echo the salience of human visual perception and demonstrate an unappreciated capacity of the center-surround architecture to facilitate novel object detection and dynamic signal representation.

Efficient and precise release of glutamate from retinal bipolar cells is ensured by the positioning of L-type Ca(2+) channels close to release sites at the base of the synaptic ribbon. We investigated whether Ca(2+) channels at bipolar cell ribbon synapses are fixed in position or capable of moving in the membrane.