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In the retina, modulation of the amplitude of dim visual signals primarily occurs at axon terminals of rod bipolar cells (RBCs). GABA and glycine inhibitory neurotransmitter receptors and the excitatory amino acid transporter 5 (EAAT5) modulate the RBC output. EAATs clear glutamate from the synapse, but they also have a glutamate-gated chloride conductance. EAAT5 acts primarily as an inhibitory glutamate-gated chloride channel. The relative role of visually evoked EAAT5 inhibition compared with GABA and glycine inhibition has not been addressed. In this study, we determine the contribution of EAAT5-mediated inhibition onto RBCs in response to light stimuli in mouse retinal slices. We find differences and similarities in the two forms of inhibition. Our results show that GABA and glycine mediate nearly all lateral inhibition onto RBCs, as EAAT5 is solely a mediator of RBC feedback inhibition. We also find that EAAT5 and conventional GABA inhibition both contribute to feedback inhibition at all stimulus intensities. Finally, our in silico modeling compares and contrasts EAAT5-mediated to GABA- and glycine-mediated feedback inhibition. Both forms of inhibition have a substantial impact on synaptic transmission to the postsynaptic AII amacrine cell. Our results suggest that the late phase EAAT5 inhibition acts with the early phase conventional, reciprocal GABA inhibition to modulate the rod signaling pathway between rod bipolar cells and their downstream synaptic targets. NEW & NOTEWORTHY Excitatory amino acid transporter 5 (EAAT5) glutamate transporters have a chloride channel that is strongly activated by glutamate, which modulates excitatory signaling. We found that EAAT5 is a major contributor to feedback inhibition on rod bipolar cells. Inhibition to rod bipolar cells is also mediated by GABA and glycine. GABA and glycine mediate the early phase of feedback inhibition, and EAAT5 mediates a more delayed inhibition. Together, inhibitory transmitters and EAAT5 coordinate to mediate feedback inhibition, controlling neuronal output.

Inherited retinal degenerations encompass a wide range of diseases that result in the death of rod and cone photoreceptors, eventually leading to irreversible blindness. Low vision survives at early stages of degeneration, at which point it could rely on residual populations of rod/cone photoreceptors as well as the inner retinal photoreceptor, melanopsin. To date, the impact of partial retinal degeneration on visual responses in the primary visual thalamus (dorsal lateral geniculate nucleus, dLGN) remains unknown, as does their relative reliance on surviving rod and cone photoreceptors vs. melanopsin. To answer these questions, we recorded visually evoked responses in the dLGN of anesthetized rd 1 mice using in vivo electrophysiology at an age (3-5 wk) at which cones are partially degenerate and rods are absent. We found that excitatory (ON) responses to light had lower amplitude and longer latency in rd 1 mice compared with age-matched visually intact controls; however, contrast sensitivity and spatial receptive field size were largely unaffected at this early stage of degeneration. Responses were retained when those wavelengths to which melanopsin is most sensitive were depleted, indicating that they were driven primarily by surviving cones. Inhibitory responses appeared absent in the rd 1 thalamus, as did light-evoked gamma oscillations in firing. This description of fundamental features of the dLGN visual response at this intermediate stage of retinal degeneration provides a context for emerging attempts to restore vision by introducing ectopic photoreception to the degenerate retina. NEW & NOTEWORTHY This study provides new therapeutically relevant insights to visual responses in the dorsal lateral geniculate nucleus during progressive retinal degeneration. Using in vivo electrophysiology, we demonstrate that visual responses have lower amplitude and longer latency during degeneration, but contrast sensitivity and spatial receptive fields remain unaffected. Such visual responses are driven predominantly by surviving cones rather than melanopsin photoreceptors. The functional integrity of this visual pathway is encouraging for emerging attempts at visual restoration.

Sensory receptor noise corrupts sensory signals, contributing to imperfect perception and dictating central processing strategies. For example, noise in rod phototransduction limits our ability to detect light, and minimizing the impact of this noise requires precisely tuned nonlinear processing by the retina. But detection sensitivity is only one aspect of night vision: prompt and accurate behavior also requires that rods reliably encode the timing of photon arrivals. We show here that the temporal resolution of responses of primate rods is much finer than the duration of the light response and identify the key limiting sources of transduction noise. We also find that the thermal activation rate of rhodopsin is lower than previous estimates, implying that other noise sources are more important than previously appreciated. A model of rod single-photon responses reveals that the limiting noise relevant for behavior depends critically on how rod signals are pooled by downstream neurons. NEW & NOTEWORTHY Many studies have focused on the visual system's ability to detect photons, but not on its ability to encode the relative timing of detected photons. Timing is essential for computations such as determining the velocity of moving objects. Here we examine the timing precision of primate rod photoreceptor responses and show that it is more precise than previously appreciated. This motivates an evaluation of whether scotopic vision approaches limits imposed by rod temporal resolution.

Dopamine modulation of retinal signaling has been shown to be an important part of retinal adaptation to increased background light levels, but the role of dopamine modulation of retinal inhibition is not clear. We previously showed that light adaptation causes a large reduction in inhibition to rod bipolar cells, potentially to match the decrease in excitation after rod saturation. In this study, we determined how dopamine D1 receptors in the inner retina contribute to this modulation. We found that D1 receptor activation significantly decreased the magnitude of inhibitory light responses from rod bipolar cells, whereas D1 receptor blockade during light adaptation partially prevented this decline. To determine what mechanisms were involved in the modulation of inhibitory light responses, we measured the effect of D1 receptor activation on spontaneous currents and currents evoked from electrically stimulating amacrine cell inputs to rod bipolar cells. D1 receptor activation decreased the frequency of spontaneous inhibition with no change in event amplitudes, suggesting a presynaptic change in amacrine cell activity in agreement with previous reports that rod bipolar cells lack D1 receptors. Additionally, we found that D1 receptor activation reduced the amplitude of electrically evoked responses, showing that D1 receptors can modulate amacrine cells directly. Our results suggest that D1 receptor activation can replicate a large portion but not all of the effects of light adaptation, likely by modulating release from amacrine cells onto rod bipolar cells. NEW & NOTEWORTHY We demonstrated a new aspect of dopaminergic signaling that is involved in mediating light adaptation of retinal inhibition. This D1 receptor-dependent mechanism likely acts through receptors located directly on amacrine cells, in addition to its potential role in modulating the strength of serial inhibition between amacrine cells. Our results also suggest that another D2/D4 receptor-dependent or dopamine-independent mechanism must also be involved in light adaptation of inhibition to rod bipolar cells.

We have determined the impact of rod death and cone reorganization on the spatiotemporal receptive fields (RFs) and spontaneous activity of distinct retinal ganglion cell (RGC) types. We compared RGC function between healthy and retinitis pigmentosa (RP) model rats (S334ter-3) at a time when nearly all rods were lost but cones remained. This allowed us to determine the impact of rod death on cone-mediated visual signaling, a relevant time point because the diagnosis of RP frequently occurs when patients are nightblind but daytime vision persists. Following rod death, functionally distinct RGC types persisted; this indicates that parallel processing of visual input remained largely intact. However, some properties of cone-mediated responses were altered ubiquitously across RGC types, such as prolonged temporal integration and reduced spatial RF area. Other properties changed in a cell type-specific manner, such as temporal RF shape (dynamics), spontaneous activity, and direction selectivity. These observations identify the extent of functional remodeling in the retina following rod death but before cone loss. They also indicate new potential challenges to restoring normal vision by replacing lost rod photoreceptors. NEW & NOTEWORTHY This study provides novel and therapeutically relevant insights to retinal function following rod death but before cone death. To determine changes in retinal output, we used a large-scale multielectrode array to simultaneously record from hundreds of retinal ganglion cells (RGCs). These recordings of large-scale neural activity revealed that following the death of all rods, functionally distinct RGCs remain. However, the receptive field properties and spontaneous activity of these RGCs are altered in a cell type-specific manner., (Copyright © 2017 the American Physiological Society.)

At many glutamatergic synapses, non-N-methyl-d-aspartate (NMDA) and NMDA receptors are coexpressed postsynaptically. In the mammalian retina, glutamatergic rod bipolar cells are presynaptic to two rod amacrine cells (AII and A17) that constitute dyad postsynaptic partners opposite each presynaptic active zone. Whereas there is strong evidence for expression of non-NMDA receptors by both AII and A17 amacrines, the expression of NMDA receptors by the pre- and postsynaptic neurons in this microcircuit has not been resolved. In this study, using patch-clamp recording from visually identified cells in rat retinal slices, we investigated the expression and functional properties of NMDA receptors in these cells with a combination of pharmacological and biophysical methods. Pressure application of NMDA did not evoke a response in rod bipolar cells, but for both AII and A17 amacrines, NMDA evoked responses that were blocked by a competitive antagonist (CPP) applied extracellularly and an open channel blocker (MK-801) applied intracellularly. NMDA-evoked responses also displayed strong Mg(2+)-dependent voltage block and were independent of gap junction coupling. With low-frequency application (60-s intervals), NMDA-evoked responses remained stable for up to 50 min, but with higher-frequency stimulation (10- to 20-s intervals), NMDA responses were strongly and reversibly suppressed. We observed strong potentiation when NMDA was applied in nominally Ca(2+)-free extracellular solution, potentially reflecting Ca(2+)-dependent NMDA receptor inactivation. These results indicate that expression of functional (i.e., conductance-increasing) NMDA receptors is common to both AII and A17 amacrine cells and suggest that these receptors could play an important role for synaptic signaling, integration, or plasticity in the rod pathway., (Copyright © 2016 the American Physiological Society.)

The mouse is commonly used for studying retinal processing, primarily because it is amenable to genetic manipulation. To accurately study photoreceptor driven signals in the healthy and diseased retina, it is of great importance to isolate the responses of single photoreceptor types. This is not easily achieved in mice because of the strong overlap of rod and M-cone absorption spectra (i.e., maxima at 498 and 508 nm, respectively). With a newly developed mouse model (Opn1lw(LIAIS)) expressing a variant of the human L-cone pigment (561 nm) instead of the mouse M-opsin, the absorption spectra are substantially separated, allowing retinal physiology to be studied using silent substitution stimuli. Unlike conventional chromatic isolation methods, this spectral compensation approach can isolate single photoreceptor subtypes without changing the retinal adaptation. We measured flicker electroretinograms in these mutants under ketamine-xylazine sedation with double silent substitution (silent S-cone and either rod or M/L-cones) and obtained robust responses for both rods and (L-)cones. Small signals were yielded in wild-type mice, whereas heterozygotes exhibited responses that were generally intermediate to both. Fundamental response amplitudes and phase behaviors (as a function of temporal frequency) in all genotypes were largely similar. Surprisingly, isolated (L-)cone and rod response properties in the mutant strain were alike. Thus the LIAIS mouse warrants a more comprehensive in vivo assessment of photoreceptor subtype-specific physiology, because it overcomes the hindrance of overlapping spectral sensitivities present in the normal mouse., (Copyright © 2015 the American Physiological Society.)

In advanced retinal degeneration loss of rods and cones leaves melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) as the only source of visual information. ipRGCs drive non-image-forming responses (e.g., circadian photoentrainment) under such conditions but, despite projecting to the primary visual thalamus [dorsal lateral geniculate nucleus (dLGN)], do not support form vision. We wished to determine what precludes ipRGCs supporting spatial discrimination after photoreceptor loss, using a mouse model (rd/rd cl) lacking rods and cones. Using multielectrode arrays, we found that both RGCs and neurons in the dLGN of this animal have clearly delineated spatial receptive fields. In the retina, they are typically symmetrical, lack inhibitory surrounds, and have diameters in the range of 10-30° of visual space. Receptive fields in the dLGN were larger (diameters typically 30-70°) but matched the retinotopic map of the mouse dLGN. Injections of a neuroanatomical tracer (cholera toxin β-subunit) into the dLGN confirmed that retinotopic order of ganglion cell projections to the dLGN and thalamic projections to the cortex is at least superficially intact in rd/rd cl mice. However, as previously reported for deafferented ipRGCs, onset and offset of light responses have long latencies in the rd/rd cl retina and dLGN. Accordingly, dLGN neurons failed to track dynamic changes in light intensity in this animal. Our data reveal that ipRGCs can convey spatial information in advanced retinal degeneration and identify their poor temporal fidelity as the major limitation in their ability to provide information about spatial patterns under natural viewing conditions., (Copyright © 2015 the American Physiological Society.)

The retina responds to a wide range of light stimuli by adaptation of retinal signaling to background light intensity and the use of two different photoreceptors: rods that sense dim light and cones that sense bright light. Rods signal to rod bipolar cells that receive significant inhibition from amacrine cells in the dark, especially from a rod bipolar cell-activated GABAergic amacrine cell. This inhibition modulates the output of rod bipolar cells onto downstream neurons. However, it was not clear how the inhibition of rod bipolar cells changes when rod signaling is limited by an adapting background light and cone signaling becomes dominant. We found that both light-evoked and spontaneous rod bipolar cell inhibition significantly decrease with light adaptation. This suggests a global decrease in the activity of amacrine cells that provide input to rod bipolar cells with light adaptation. However, inhibition to rod bipolar cells is also limited by GABAergic connections between amacrine cells, which decrease GABAergic input to rod bipolar cells. When we removed this serial inhibition, the light-evoked inhibition to rod bipolar cells remained after light adaptation. These results suggest that decreased inhibition to rod bipolar cells after light adaptation is due to decreased rod pathway activity as well as an active increase in inhibition between amacrine cells. Together these serve to limit rod bipolar cell inhibition after light adaptation, when the rod pathway is inactive and modulation of the signal is not required. This suggests an efficiency mechanism in the retina to limit unnecessary signaling.

Ca(2+) signaling in synaptic terminals plays a critical role in neurotransmitter release and short-term synaptic plasticity. In the present study, we examined the role of synaptic Ca(2+) handling mechanisms in the synaptic terminals of mammalian rod bipolar cells, neurons that play a pivotal role in the high-sensitivity vision pathway. We found that mitochondria sequester Ca(2+) under conditions of high Ca(2+) load, maintaining intraterminal Ca(2+) near resting levels. Indeed, the effect of the mitochondria was so powerful that the ability to clamp intraterminal Ca(2+) with a somatically positioned whole cell patch pipette was compromised. The plasma membrane Ca(2+)-ATPase (PMCA), but not the Na(+)/Ca(2+) exchanger (NCX) or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), was an important regulator of resting Ca(2+). Furthermore, PMCA activity, but not NCX or SERCA activity, was essential for the recovery of Ca(2+) levels following depolarization-evoked Ca(2+) entry. Loss of PMCA function was also associated with impaired restoration of membrane surface area following depolarization-evoked exocytosis. Given its roles in the regulation of intraterminal Ca(2+) at rest and after a stimulus-evoked Ca(2+) rise, the PMCA is poised to modulate luminance coding and adaptation to background illumination in the mammalian rod bipolar cell.

Life on earth is subject to alternating cycles of day and night imposed by the rotation of the earth. Consequently, living things have evolved photodetective systems to synchronize their physiology and behavior with the external light-dark cycle. This form of photodetection is unlike the familiar "image vision," in that the basic information is light or darkness over time, independent of spatial patterns. "Nonimage" vision is probably far more ancient than image vision and is widespread in living species. For mammals, it has long been assumed that the photoreceptors for nonimage vision are also the textbook rods and cones. However, recent years have witnessed the discovery of a small population of retinal ganglion cells in the mammalian eye that express a unique visual pigment called melanopsin. These ganglion cells are intrinsically photosensitive and drive a variety of nonimage visual functions. In addition to being photoreceptors themselves, they also constitute the major conduit for rod and cone signals to the brain for nonimage visual functions such as circadian photoentrainment and the pupillary light reflex. Here we review what is known about these novel mammalian photoreceptors.

While connections between inhibitory interneurons are common circuit elements, it has been difficult to define their signal processing roles because of the inability to activate these circuits using natural stimuli. We overcame this limitation by studying connections between inhibitory amacrine cells in the retina. These interneurons form spatially extensive inhibitory networks that shape signaling between bipolar cell relay neurons to ganglion cell output neurons. We investigated how amacrine cell networks modulate these retinal signals by selectively activating the networks with spatially defined light stimuli. The roles of amacrine cell networks were assessed by recording their inhibitory synaptic outputs in bipolar cells that suppress bipolar cell output to ganglion cells. When the amacrine cell network was activated by large light stimuli, the inhibitory connections between amacrine cells unexpectedly depressed bipolar cell inhibition. Bipolar cell inhibition elicited by smaller light stimuli or electrically activated feedback inhibition was not suppressed because these stimuli did not activate the connections between amacrine cells. Thus the activation of amacrine cell circuits with large light stimuli can shape the spatial sensitivity of the retina by limiting the spatial extent of bipolar cell inhibition. Because inner retinal inhibition contributes to ganglion cell surround inhibition, in part, by controlling input from bipolar cells, these connections may refine the spatial properties of the retinal output. This functional role of interneuron connections may be repeated throughout the CNS.

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.

To meet the high-energy demands of photoreceptor cells, the outer retina metabolizes glucose through glycolytic and oxidative pathways, resulting in large-scale production of lactate and CO(2). Mct3, a proton-coupled monocarboxylate transporter, is critically positioned to facilitate transport of lactate and H(+) out of the retina and could therefore play a role in pH and ion homeostasis of the outer retina. Mct3 is preferentially expressed in the basolateral membrane of the retinal pigment epithelium and forms a heteromeric complex with the accessory protein CD147. To examine the physiological role of Mct3 in the retina, we generated mice with a targeted deletion in Mct3 (slc16A8). The overall retinal histology of 4- to 36-wk-old Mct3(-/-) mice appeared normal. In the absence of Mct3, expression of CD147 was lost from the basolateral but not apical RPE. The saturated a-wave amplitude (a(max)) of the scotopic electroretinogram (ERG) was reduced by approximately twofold in Mct3(-/-) mice relative to wild-type mice. A fourfold increase in lactate in the retina suggested a decrease in outer-retinal pH. In single-cell recordings from superfused retinal slices, saturating amplitudes of single rod photocurrents (J(max)) were comparable indicating that Mct3(-/-) mouse photoreceptor cells were inherently healthy. Based on these data, we hypothesize that disruption of Mct3 leads to a potentially reversible decrease in subretinal space pH, thereby reducing the magnitude of the light suppressible photoreceptor current.

Retinal bipolar cells can be divided into on and off types based on the polarity of their response to light. Bipolar activity is further shaped by inhibitory inputs, characterized here by the events that occur immediately after the onset of a light step: 1) in most off bipolar cells, excitatory current decreased, whereas inhibitory current increased. These currents reinforced each other, enhancing the light response. 2) In about half of the on cone bipolar cells, the excitatory current increased, whereas inhibitory current decreased, also reinforcing the light response. Both of these reinforcing interactions were mediated by glycinergic inhibition. 3) In the remaining on cone bipolar cells, excitation and inhibition both increased, but inhibition was delayed so that these cells responded transiently. 4) Finally, in rod bipolar cells, excitation and inhibition both increased so that inhibition suppressed excitation, reducing the light response at all time scales. The suppressive inhibition seen in on cone and rod bipolar cells was mediated by GABA. Thus morphologically diverse bipolar cells receive only four main types of inhibitory input, and the majority of "inhibitory" inputs actually serve to enhance excitation.

To extend the usefulness of rod bipolar cells for studies of chemical synaptic transmission, we have performed electrophysiological recordings from rod bipolar axon terminals in an in vitro slice preparation of the rat retina. Whole cell recordings from axon terminals and cell bodies were used to investigate the passive membrane properties of rod bipolar cells and analyzed with a two-compartment equivalent electrical circuit model developed by Mennerick et al. For both terminal- and soma-end recordings, capacitive current decays were well fitted by biexponential functions. Computer simulations of simplified models of rod bipolar cells demonstrated that estimates of the capacitance of the axon terminal compartment can depend critically on the recording location, with terminal-end recordings giving the best estimates. Computer simulations and whole cell recordings demonstrated that terminal-end recordings can yield more accurate estimates of the peak amplitude and kinetic properties of postsynaptic currents generated at the axon terminals due to increased electrotonic filtering of these currents when recorded at the soma. Finally, we present whole cell and outside-out patch recordings from axon terminals with responses evoked by GABA and glycine, spontaneous inhibitory postsynaptic currents, voltage-gated Ca(2+) currents, and depolarization-evoked reciprocal synaptic responses, verifying that the recorded axon terminals are involved in normal pre- and postsynaptic relationships. These results demonstrate that axon terminals of rod bipolar cells are directly accessible to whole cell and outside-out patch recordings, extending the usefulness of this preparation for detailed studies of pre- and postsynaptic mechanisms of synaptic transmission in the CNS.

The mouse is an important model system for understanding the molecular basis of neuronal signaling and diseases of synaptic communication. However, the best-characterized retinal ribbon-style synapses are those of nonmammalian vertebrates. To remedy this situation, we asked whether it would be feasible to track synaptic vesicle dynamics in the isolated mouse rod bipolar cell using time-resolved capacitance measurements. The results demonstrate that membrane depolarization triggered an increase in membrane capacitance that was Ca(2+) dependent and restricted to the synaptic compartment, consistent with exocytosis. The amplitude of the capacitance response recorded from the easily accessible soma of an intact mouse rod bipolar cell was identical to that recorded directly from the small synaptic terminal, suggesting that in the carefully selected cohort of cells presented here, axonal resistance was not a significant barrier to current flow. This supposition was supported by the analysis of passive membrane properties and a comparison of membrane capacitance measurements in cells with and without synaptic terminals and reinforced by the lack of an effect of sine-wave frequency (200-1,600 Hz) on the measured capacitance increase. The magnitude of the capacitance response increased with Ca(2+) entry until a plateau was reached at a spatially averaged intraterminal calcium of about 600 nM. We interpret this plateau, nominally 30 fF, as corresponding to a releasable pool of synaptic vesicles. The robustness of this measure suggests that capacitance measurements may be used in the mouse rod bipolar cell to compare pool size across treatment conditions.

Rod signals traverse several synapses en route to cone bipolar cells. In one pathway, rods communicate directly with cones via gap junctions. In a second pathway, signals flow rods-rod bipolars-AII amacrines-cone bipolars. The relative contribution of each pathway to retinal function is not well understood. Here we have examined this question from the perspective of the AII amacrine. AIIs form bidirectional electrical synapses with on cone bipolars. Consequently, as on cone bipolars are activated by outer plexiform inputs, they too should contribute to the AII response. Rod bipolar inputs to AIIs were blocked by AMPA receptor antagonists, revealing a smaller, non-AMPA component of the light response. This small residual response did not reverse between -70 and +70 mV and was blocked by carbenoxolone, suggesting that the current arose in on cone bipolars and was transmitted to AIIs via gap junctions. The residual component was evident for stimuli 2 log units below cone threshold and was prolonged for bright stimuli, demonstrating that it was rod driven. Because the rod bipolar-AII pathway was blocked, the rod-driven residual current likely was generated via the rod-cone pathway activation of on cone bipolars. Thus for a large range of intensities, rod signals reach the inner retina by both rod bipolar-AII and rod-cone coupling pathways.

Recent reports on rod photoreceptor neuroprotection by Ca2+ channel blockers have pointed out the need to assess the effect of these blockers on mammalian rods. However, in mammals, rod electrophysiological characterization has been hampered by the small size of these photoreceptors, which were instead extensively studied in nonmammalian vertebrates. To further characterize ionic conductances and to assess the pharmacology of Ca2+ channels in mammalian rods, freshly dissociated pig rod photoreceptors were recorded with the whole cell patch-clamp technique. Rod cells expressed 1) a hyperpolarization-activated inward-rectifying conductance (I(h)) sensitive to external Cs+; 2) a sustained outward K+ current (I(K)) sensitive to tetraethylammonium; 3) a sustained voltage-gated Ca2+ current (I(Ca)) sensitive to benzothiazepine (diltiazem) and phenylalkylamine (verapamil) derivatives; 4) a Ca(2+)-activated Cl- current (I(Cl(Ca))); and 5) a plasma membrane Ca(2+)-ATPase. The Ca2+ current showed a range of activation from positive potentials to -60 mV with a maximum between -30 and -20 mV. In contrast to other L-type Ca2+ channels, rod Ca2+ channels were blocked at similar and relatively high concentrations by the diltiazem isomers and verapamil. The biphasic dose-response for D-diltiazem confirmed the low sensitivity of Ca2+ channels for the molecule. The ATPase, which was localized at the axon terminal, was found to contribute to Ca2+ extrusion. These results suggest that the electrophysiological features of rod photoreceptors had been preserved during evolution from nonmammalian vertebrates to mammals. This work indicates further that mammalian rods express nonclassic L-type Ca2+ channels, showing a low sensitivity to the diltiazem isomers used in neuroprotective studies.

Glutamate receptors on giant danio retinal on bipolar cells were studied with whole cell patch clamping using a slice preparation. Cone-driven on bipolars (Cbs) and mixed-input on bipolars (Mbs) were identified morphologically. Most Cbs responded to the excitatory amino acid transporter (EAAT) substrate d-aspartate but not to the group III metabotropic glutamate receptor (mGluR) agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) or the AMPA/kainate receptor agonist kainate, suggesting EAATs are the primary glutamate receptors on Cbs. The EAAT inhibitor dl-threo-beta-benzyloxyasparate (TBOA) blocked all light-evoked responses of Cbs, suggesting these responses are mediated exclusively by EAATs. Conversely, all Mbs responded to d-aspartate and l-AP4 but not to kainate, indicating they have both EAATs and group III mGluRs (presumably mGluR6). The light responses of Mbs involve both receptors because they could be blocked by TBOA plus (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III mGluR antagonist) but not by either alone. Under dark-adapted conditions, the responses of Mbs to green (rod-selective) stimuli were reduced by CPPG but enhanced by TBOA. In contrast, both antagonists reduced the responses to red (cone-selective) stimuli, although TBOA was more effective. Furthermore, under photopic conditions, TBOA failed to eliminate light-evoked responses of Mbs. Thus on Mbs, rod inputs are mediated predominantly by mGluR6, whereas cone inputs are mediated mainly by EAATs but also by mGluR6 to some extent. Finally, we explored the interactions between EAATs and mGluR6 in Mbs. Responses to d-aspartate were reduced by l-AP4 and vice versa. Therefore mGluR6 and EAATs suppress each other, and this might underlie mutual suppression between rod and cone signals in Mbs.

Unlabelled: Electroretinograms (ERGs) were recorded from the giant danio (Danio aequipinnatus) to study glutamatergic input mechanisms onto bipolar cells. Glutamate analogs were applied to determine which receptor types mediate synaptic transmission from rods and cones to on and off bipolar cells. Picrotoxin, strychnine, and tetrodotoxin were used to isolate the effects of the glutamate analogs to the photoreceptor-bipolar cell synapse. Under photopic conditions, the group III metabotropic glutamate receptor (mGluR) antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) only slightly reduced the b-wave, whereas the excitatory amino acid transporter (EAAT) blocker dl-threo-beta-benzyl-oxyaspartate (TBOA) removed most of it. Complete elimination of the b-wave required both antagonists. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) blocked the d-wave. Under scotopic conditions, rod and cone inputs onto on bipolar cells were studied by comparing the sensitivities of the b-wave to photopically matched green and red stimuli. The b-wave was >1 log unit more sensitive to the green than to the red stimulus under control conditions. In CPPG or l-AP4 (l-(+)-2-amino-4-phosphonobutyric acid, a group III mGluR agonist), the sensitivity of the b-wave to the green stimulus was dramatically reduced and the b-waves elicited by the 2 stimuli became nearly matched. The d-wave elicited by dim green stimuli, which presumably could be detected only by the rods, was eliminated by NBQX., In Conclusion: 1) cone signals onto on bipolar cells involve mainly EAATs but also mGluRs (presumably mGluR6) to a lesser extent; 2) rods signal onto on bipolars by mainly mGluR6; 3) off bipolar cells receive signals from both photoreceptor types by AMPA/kainate receptors.

Circadian clocks are integral components of visual systems. They help adjust an animal's vision to diurnal changes in ambient illumination. To understand how circadian clocks may adapt visual sensitivity, we investigated the spatial and temporal properties of optomotor responses of young Xenopus laevis tadpoles (Nieuwkoop and Faber, developmental stage 48) using a modified 2-alternative preferential-viewing method. We maintained animals in constant darkness and measured temporal sensitivity during their subjective day and night. We found that their behavioral responses can be explained in terms of 2 mechanisms with different temporal properties. The more sensitive mechanism operates at low temporal frequencies and intermediate wavelengths (lambdamax = 520 nm), properties consistent with rod signals. Threshold for this mechanism is approximately 0.04 photoisomerizations rod(-1) s(-1), consistent with single-photon detection. A less-sensitive mechanism responds to higher temporal frequencies (cutoff = 12 Hz) and has broad spectral sensitivity (370-720 nm), consistent with multiple classes of cone signals. This cone mechanism does not change, but the cutoff frequency of the more sensitive rod mechanism shifts from 0.35 Hz at night to 1.1 Hz during the subjective day, thereby enhancing the animal's sensitivity to dim rapidly changing stimuli. This day-night shift in rod temporal cutoff frequency cycles in complete darkness, characteristic of an endogenous circadian rhythm. The temporal properties of the behaviorally measured rod mechanism correspond closely with those of the electrophysiologically measured retinal response, indicating that the rod signals are modulated at the level of the outer retina.

The abundance of nitric oxide (NO) synthesizing enzymes identified in the vertebrate retina highlight the importance of NO as a signaling molecule in this tissue. Here we describe opposing actions of NO on the rod and cone photoreceptor synapse. Depolarization-induced increases of calcium concentration in rods and cones were enhanced and inhibited, respectively, by the NO donor S-nitrosocysteine. NO suppressed calcium current in cones by decreasing the maximum conductance, whereas NO facilitated rod Ca channel activation. NO also activated a nonselective voltage-independent conductance in both rods and cones. Suppression of NO production in the intact retina with N(G)-nitro-l-arginine favored cone over rod driven postsynaptic signals, as would be expected if NO enhanced rod and suppressed cone synaptic activity. These findings may imply involvement of NO in regulating the strength of rod and cone pathways in the retina during different states of adaptation.

In response to light, the retinal pigment epithelium (RPE) generates a series of slow potentials that can be recorded as the c-wave, fast oscillation (FO), and light peak (LP) of the electroretinogram (ERG). As these potentials can be related to specific cellular events, they provide information about RPE function and how that may be altered by disease or experimental manipulation. In the present study we describe a noninvasive means for recording the light-evoked responses of the mouse RPE and use this to define the stimulus-response properties of the major components in three inbred strains of mice (BALBc/ByJ, C57BL/6J, and 129/SvJ) and two mouse mutants that reduce activity in the rod pathway. All of the major ERG components generated by the RPE are readily measured in the mouse. In albino strains (BALBc/ByJ and 129/SvJ) the intensity-response functions for the c-wave, FO, and LP are shifted toward lower intensities in comparison to those for C57BL/6J mice. Each of these components was markedly reduced in mice lacking transducin in which rod phototransduction is interrupted, indicating that they reflect primarily rod photoreceptor activity. All components were observed in no b-wave (nob) mutant mice, indicating that inner retinal activity does not make a major contribution to these potentials. Further studies of mutant mice will allow us to define the functional consequences of gene manipulation on RPE function and to evaluate specific hypotheses regarding the generation of ERG components.

Several lines of evidence suggest that nitric oxide (NO) can regulate diverse retinal functions, but whether this gas is capable of modulating the visual responses of retinal output neurons has not been established. In the present study the effects of NO on rod-driven responses of retinal ganglion cells were tested by making whole cell patch-clamp recordings from morphologically identified ganglion cells in the isolated ferret retina. Bath application of L-arginine, the substrate of nitric oxide synthase, and S-nitroso-N-acetylpenicillamine, the NO donor, was found to differentially affect on and off discharge patterns. The introduction of these drugs significantly decreased visual responses of retinal ganglion cells, but the effects were more pronounced on off than on on discharges. The peak discharge rates of on responses were usually reduced by about 40%, but not completely blocked. In contrast, off responses were completely blocked in most cells. These differential effects were observed in on-off cells as well as in cells that yielded just on or off discharges. The off responses that were blocked by NO were also blocked by DL-2-amino-phosphonobutyric acid (APB) and strychnine, suggesting the involvement of the APB-sensitive rod pathway.

Adenosine is released from retina in darkness; photoreceptors possess A2 adenosine receptors, and A2 agonists inhibit L-type Ca2+ currents (ICa) in rods. We therefore investigated whether A2 agonists inhibit rod inputs into second-order neurons and whether selective antagonists to A1, A2A, or A3 receptors prevent Ca2+ influx through rod ICa. [Ca2+]i changes in rods were assessed with fura-2. ICa in rods and light responses of rods and second-order neurons were recorded using perforated patch-clamp techniques in the aquatic tiger salamander retinal slice preparation. Consistent with earlier results using the A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), the A2A agonist CGS-21680 significantly inhibited ICa and depolarization-evoked [Ca2+]i increases in rods. The A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and A2A antagonist, ZM-241385, but not the A3 antagonist, VUF-5574, inhibited effects of adenosine on Ca2+ influx in rods. DPCPX and ZM-241385 also inhibited effects of CGS-21680, suggesting they both act at A2A receptors. Both A2 agonists, CGS-21680 and DPMA, reduced light-evoked currents in second-order neurons but not light-evoked voltage responses of rods, suggesting that activation of A2 receptors inhibits transmitter release from rods. The inhibitory effects of CGS-21680 on both depolarization-evoked Ca2+ influx and light-evoked currents in second-order neurons were antagonized by ZM-241385. By itself, ZM-241385 enhanced the light-evoked currents in second-order neurons, suggesting that endogenous levels of adenosine inhibit transmitter release from rods. The effects of these drugs suggest that endogenous adenosine activates an A2-like adenosine receptor on rods leading to inhibition of ICa, which in turn inhibits l-glutamate release from rod photoreceptors.

Synaptic transmission at the photoreceptor synapse is characterized by continuous release of glutamate in darkness. Release is regulated by the intracellular calcium concentration ([Ca2+]i). We here examined the physiological properties of exocytosis in tiger salamander (Ambystoma tigrinum) retinal rods and cones. Patch-clamp capacitance measurements were used to monitor exocytosis elicited by a rapid and uniform increase in [Ca2+]i by photolysis of the caged Ca2+ compound NP-EGTA. The amplitude of flash-induced increases in membrane capacitance (Cm) varied monotonically with [Ca2+]i beyond approximately 15 microM. The following two types of kinetic responses in Cm were recorded in both rods and cones: 1) a single exponential rise (39% of cells) or 2) a double-exponential rise (61%). Average rate constants of rapid and slow exocytotic responses were 420 +/- 168 and 7.85 +/- 5.02 s-1, respectively. The rate constant for the single exponential exocytotic response was 17.5 +/- 12.4 s-1, not significantly different from that of the slow exocytotic response. Beyond the threshold [Ca2+]i of approximately 15 microM, the average amplitude of rapid, slow, and single Cm response were 0.84 +/- 0.35, 0.82 +/- 0.20, and 0.70 +/- 0.23 pF, respectively. Antibodies against synaptotagmin I, a vesicle protein associated with fast exocytosis, strongly stained the synaptic terminal of isolated photoreceptors, suggesting the presence of fusion-competent vesicles. Our results confirm that photoreceptors possess a large rapidly releasable pool activated by a low-affinity Ca2+ sensor whose kinetic and calcium-dependent properties are similar to those reported in retinal bipolar cells and cochlear hair cells.

Cannabinoid CB1 receptors have been detected in retinas of numerous species, with prominent labeling in photoreceptor terminals of the chick and monkey. CB1 labeling is well-conserved across species, suggesting that CB1 receptors might also be present in photoreceptors of the tiger salamander. Synaptic transmission in vertebrate photoreceptors is mediated by L-type calcium currents-currents that are modulated by CB1 receptors in bipolar cells of the tiger salamander. Presence of CB1 receptors in photoreceptor terminals would therefore be consistent with presynaptic modulation of synaptic transmission, a role seen for cannabinoids in other parts of the brain. Here we report immunohistochemical and electrophysiological evidence for the presence of functional CB1 receptors in rod and cone photoreceptors of the tiger salamander. The cannabinoid receptor agonist WIN 55212-2 enhances calcium currents of rod photoreceptors by 39% but decreases calcium currents of large single cones by 50%. In addition, WIN 55212-2 suppresses potassium currents of rods and large single cones by 44 and 48%, respectively. Thus functional CB1 receptors, present in the terminals of rod and cone photoreceptors, differentially modulate calcium and potassium currents in rods and large single cones. CB1 receptors are therefore well positioned to modulate neurotransmitter release at the first synapse of the visual system.

We examined function of the feedback pathway from A17 GABAergic amacrine cells to rod bipolar cells (A17 feedback), a critically located inhibitory circuit in the classic rod pathway of the mammalian retina whose role in processing of scotopic visual information is still poorly understood. We show evidence that this A17 feedback has a profound influence on the temporal properties of rod-driven postphotoreceptoral responses (assessed with the scotopic electroretinogram b-wave). Application of a GABA(c) antagonist prolonged preferentially the decay of the scotopic b-wave. The degree of prolongation increased as the light intensity decreased. Application of selective GABA(a) antagonists accelerated the kinetics of the scotopic b-wave. This effect was abolished when the GABA(c) antagonist was coapplied. Selective ablation of A17 cells mimicked the action of the GABA(c) antagonist. In A17 cell-ablated retinas, the GABA(c) antagonist was no longer very effective to slow the decay of the scotopic b-wave. Thus the A17 feedback, activated by light stimulation and mediated mainly by the GABA(c) receptors, makes the scotopic b-wave more transient by accelerating preferentially its decay. The strength of the feedback can be modulated by GABA(a) receptor-mediated inhibition and by light intensity. Our results also suggest that in the mammalian retina the feedback may be a novel mechanism that contributes postphotoreceptorally to the termination of rod signals, especially those elicited by very dim light stimuli.

Depolarization-induced facilitation of L-type Ca channels in rod photoreceptors was investigated with nystatin-perforated and ruptured whole cell patch-clamp techniques in cells isolated from tiger salamander retina. Induction of facilitation was voltage dependent with a half-maximal effect seen at prepulse potentials near +31 mV. Reversal of facilitation was time dependent with fast (tau approximately 20 ms) and slow (tau approximately 1 s) components at -60 mV. Incubation of cells with pertussis toxin or intracellular administration of guanosine 5'-O-(3-thiotriphosphate) or guanosine 5'-O-(2-thiodiphosphate) had no effect on the degree to which facilitation could be evoked, implying the absence of a significant role for G proteins. Application of the phosphatase inhibitor okadaic acid or inclusion of ATP, to boost levels of phosphorylation, or inclusion of 5'adenylylimidophosphate or inhibitors of protein kinase in the pipette, to reduce levels of phosphorylation, had no effect on the development of facilitation, suggesting that phosphorylation has little or no role in this phenomenon. These results show that the L-type Ca channels in rod photoreceptors, which appear to be composed of alpha(1F)-like subunits, undergo voltage-dependent facilitation in a manner that differs from some other L-type Ca channels which undergo facilitation via phosphorylation or through G-protein-mediated inhibition.

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.

Whole cell voltage-clamp recordings were performed to investigate voltage-activated Ca(2+) currents in acutely isolated retinal bipolar cells of rats. Two groups of morphologically different bipolar cells were observed. Bipolar cells of the first group, which represent the majority of isolated bipolar cells, were immunoreactive to protein kinase C (PKC) and, therefore likely to be rod bipolar cells. Bipolar cells of the second group, which represent only a small population of isolated bipolar cells, did not show PKC immunoreactivity and were likely to be cone bipolar cells. The validity of morphological identification of bipolar cells was further confirmed by the presence of GABA(C) responses in these cells. Bipolar cells of both groups displayed low-voltage-activated (LVA) Ca(2+) currents with similar voltage dependence of activation and steady-state inactivation. However, the activation, inactivation, and deactivation kinetics of the LVA Ca(2+) currents between rod and cone bipolar cells differed. Particularly, the LVA Ca(2+) currents of rod bipolar cells displayed both transient and sustained components. In contrast, the LVA Ca(2+) currents of cone bipolar cells were mainly transient. In addition, the LVA Ca(2+) channels of rod bipolar cells were more permeable to Ba(2+) than to Ca(2+), whereas those of cone bipolar cells were equally or less permeable to Ba(2+) than to Ca(2+). The LVA Ca(2+) currents of both rod and cone bipolar cells were antagonized by high concentrations of nimodipine with IC(50) of 17 and 23 microM, respectively, but largely resistant to Cd(2+) and Ni(2+). Bipolar cells of both groups also displayed high-voltage-activated (HVA) Ca(2+) currents. The HVA Ca(2+) currents were, at least in part, to be L-type that were potentiated by BayK-8644 (1 microM) and largely antagonized by low concentrations of nimodipine (5 microM). The L-type Ca(2+) channels were almost exclusively located at the axon terminals of rod bipolar cells but expressed at least in the cell soma of cone bipolar cells. Results of this study indicate that rod and cone bipolar cells of the mammalian retina differentially express at least two types of LVA Ca(2+) channels. Rod and cone bipolar cells also show different spatial distribution of L-type Ca(2+) channels.

Reciprocal synaptic transmission between rod bipolar cells and presumed A17 amacrine cells was studied by whole cell voltage-clamp recording of rod bipolar cells in a rat retinal slice preparation. Depolarization of a rod bipolar cell evoked two identifiable types of Ca2+ current, a T-type current that activated at about -70 mV and a current with L-type pharmacology that activated at about -50 mV. Depolarization to greater than or equal to -50 mV also evoked an increase in the frequency of postsynaptic currents (PSCs). The PSCs reversed at approximately ECl (the chloride equilibrium potential), followed changes in ECl, and were blocked by gamma-aminobutyric acidA (GABAA) and GABAC receptor antagonists and thus were identified as GABAergic inhibitory PSCs (IPSCs). Bipolar cells with cut axons displayed the T-type current but lacked an L-type current and depolarization-evoked IPSCs. Thus L-type Ca2+ channels are placed strategically at the axon terminals to mediate transmitter release from rod bipolar cells. The IPSCs were blocked by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that non-NMDA receptors mediate the feed-forward bipolar-to-amacrine excitation. The NMDA receptor antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid had no consistent effect on the depolarization-evoked IPSCs, indicating that activation of NMDA receptors is not essential for the feedforward excitation. Tetrodotoxin (a blocker of voltage-gated Na+ channels) reversibly suppressed the reciprocal response in some cells but not in others, indicating that graded potentials are sufficient for transmitter release from A17 amacrine cells, but suggesting that voltage-gated Na+ channels, under some conditions, can contribute to transmitter release.

Whole cell currents were recorded from rod and cone bipolar cells in a slice preparation of the rat retina. Use of the gramicidin D perforated-patch technique prevented loss of intracellular compounds. The recorded cells were identified morphologically by injection with Lucifer yellow. During the recordings, the cells were isolated synaptically by extracellular cobalt. To distinguish the gamma-aminobutyric acid (GABA) receptors pharmacologically, the GABAA receptor antagonist, bicuculline, and the GABAC receptor antagonist, 3-aminopropyl(methyl)phosphinic acid, were used. In all bipolar cells tested, application of GABA induced postsynaptic chloride currents that hyperpolarized the cells from their resting potential of about -40 mV. GABA was applied at different concentrations to allow for the different affinity of GABA at GABAA and GABAC receptors. At a GABA concentration of 25 microM, in the case of rod bipolar cells, approximately 70% of the current was found to be mediated by GABAC receptors. In the case of cone bipolar cells, only approximately 20% of the current was mediated by GABAC receptors. Furthermore, this GABAC-mediated fraction varied among the different morphological types of cone bipolar cells, supporting the hypothesis of distinct functional roles for the different types of cone bipolar cells. There is evidence that the efficacy of GABAC receptors is modulated by glutamate through metabotropic glutamate receptors. We tested this hypothesis by applying agonists of metabotropic glutamate receptors (mGluR)1/5 to rod bipolar cells. The specific agonist (+/-)-trans-azetidine-2, 4-dicarboxylic acid and the potent mGluR agonist quisqualic acid reduced the amplitude of the GABAC responses by 10-30%. This suggests a functional role for the modulation of GABAC receptors by the metabotropic glutamate receptors mGluR1/5.

Response sensitivity and voltage gain of the rod- and cone-bipolar cell synapses in dark-adapted tiger salamander retina. J. Neurophysiol. 78: 2662-2673, 1997. Rods, cones, and bipolar cells were recorded in superfused, flat-mounted isolated retinas of the larval tiger salamander, Ambystoma tigrinum, under dark-adapted conditions. Voltage responses of 24 rods, 15 cones, and 41 bipolar cells in dark-adapted retinas to 500 nm light steps of various intensities were listed and fitted with hyperbolic functions, and their step sensitivities and relative sensitivities (log sigma) were estimated. In the linear response-intensity ranges, the step sensitivity of rods, SS(rod), is -1.0 mV photon-1 micron2 s or 0.034 mV Rh*-1 s rod and that of the cones, SS(cone), is approximately 0. 00146 mV photon-1 micron2 s or 0.000048 mV Rh*-1 s rod. The rod and cone responses were relatively homogenous with little variations in response amplitude and sensitivity. In contrast, bipolar cell responses were heterogenous with large variations in response amplitude and sensitivity. The maximum response amplitude of bipolar cells varied from 5 to 25 mV, and the relative response sensitivity (log sigma) varied >6 log units (-8.11 to -2.32). The step sensitivity of bipolar cells in the linear response-intensity range varied from 0.0000438 to 51.82 mV photon-1 micron2 s. Bipolar cells in dark-adapted tiger salamander retinas fell into two groups according to their relative sensitivities with very few cells falling in the intermediate light intensity region. The mixed bipolar cells (DBCM and HBCM) exhibited relative response sensitivity ranged from -8.11 to -5.54, and step sensitivity ranged from 1.22 to 51.82 mV photon-1 micron2 s. The cone-driven bipolar cells (DBCC and HBCC) exhibited relative response sensitivity ranged from -3.45 to -2.32, and step sensitivity ranged from 0.0000438 to 0. 00201 mV photon-1 micron2 sec. The chord voltage gain of the rod-DBCM or rod-HBCM synapses near the rod dark membrane potential ranged from 1.14 to 48.43 and that of the cone-DBCC or cone-HBCC synaptic gain near the cone dark membrane potential ranged from 0.03 to 1.38. The highest voltage gains were found near the rod or cone dark membrane potentials. By the use of linear subtraction method, we studied the synaptic inputs from cones to five mixed bipolar cells, and the voltage gains of the cone synapses in each of the bipolar cells were very close to the voltage gain of the rod synapses. This result suggests that although the responses of mixed bipolar cells are mediated mainly by rods when lights of short and medium wavelengths are used, their responses to long wavelength lights (>650 nm) are mediated by both rods and cones with comparable synaptic gains. Functional roles of the mixed and cone-driven bipolar cells in information processing in dark-adapted retinas are discussed.

An M-like K+ current (IKx) helps set the rod photoreceptor resting potential and accelerates the response to dim light. Recorded with ruptured-patch whole cell techniques, the amplitude of IKx diminished, and activation occurred at increasingly negative potentials as a function of time. In contrast, IKx was stable during nystatin perforated-patch recording. Stability during ruptured-patch recording could be induced by raising the intracellular Ca2+ concentration ([Ca2+]i) or by including caffeine or D-myo-inositol 1,4,5-trisphosphate in the pipette. This Ca(2+)-induced stability of IKx was blocked by inhibitors of Ca2+/calmodulin-dependent protein kinases, such as W-7, KN-62, chelerythrine, or H-7. Okadaic acid, an inhibitor of protein phosphatases, maintained IKx stability even at low [Ca2+]i. The requirement for phosphorylation was demonstrated by depleting MgATP or by providing 5'-adenylylimidophosphate, a nonhydrolyzable analog of ATP, either of which blocked the Ca(2+)-induced stability of IKx. These observations show that phosphorylation regulates IKx and that a stimulus controlling this action is [Ca2+]i. Should [Ca2+]i change during light adaptation, changes in IKx might alter the resting potential and temporal response properties of rod photoreceptors.

1. In intact rods of the bullfrog, Rana Catesbeiana, that were loaded with Fura-2 by incubation, we made high-resolution measurements of Na:Ca,K exchange currents and measured cytosolic free calcium concentrations during exposure to steps of illumination. The calcium dynamics we observed are indicative of unmanipulated rods because Fura-2 had little effect on calcium buffering within the outer segment. 2. In the dark, the total concentration of calcium within the outer segment, determined by integrating the exchange current, was near 50 microM. The free calcium concentration in darkness was 200-400 nM, indicating that > of = 99% of the internal calcium was bound to buffer molecules or equivalently sequestered. 3. During saturating illumination, the concentration of free calcium near the membrane (assayed by the exchange current) fell more rapidly than the space-averaged free calcium concentration (measured with Fura-2), but both had time courses that were best described by a sum of three exponential terms. The time constants were the same for each assay, but the weighting factors were different. 4. The relationship between the exchange current and space-averaged calcium concentration is consistent with significant concentration gradients within the outer segment resulting from high buffering power, diffusional restrictions, and the fact that all net gain and loss of calcium occurs at the membrane. The data further indicate that effective buffering, and hence calcium mobility, is not uniform within the outer segment. 5. Calcium kinetics were independent of the calcium concentration, indicating that the dominant buffers effectively have a low affinity for calcium (KD >> [Ca2+]dark free). 6. The dynamics of calcium changes and of exchange currents evoked by saturating and nonsaturating illumination are completely predictable from changes in the circulating current. Calcium and current are related by a linear transformation, indicating that calcium fluxes within the outer segment are passive and that buffers equilibrate rapidly. 7. Although calcium concentrations change slowly with respect to changes in the circulating current, both measured and calculated calcium dynamics are well correlated with changes in light adaptation. Responses to test flashes depended weakly on the detailed time course of the adapting stimulus but strongly on the free cytosolic calcium concentration at the time the test flash was delivered.

1. Using the whole cell patch clamp method, we investigated the effect of dopamine on a hyperpolarization-activated current (Ih) in the inner segments of rod photoreceptors of the Xenopus retina. 2. Ih was elicited by hyperpolarizing voltage steps to -120 mV from a holding potential of -40 mV. Dopamine reversibly reduced Ih in a dose-dependent manner. Dopamine-mediated inhibition of Ih was blocked by the D2 dopamine antagonist sulpiride. 3. The D2 dopamine agonist quinpirole (0.1-20 microM) inhibited Ih whereas the D1 agonist SKF-38393 (100 microM) had no effect on Ih. Quinpirole-induced inhibition of Ih was blocked by sulpiride, but not by the D4 antagonist, clozapine. The D3 agonists (+/-)-7-hydroxy-2-dipropylaminotetralin hydrochloride and trans-7-hydroxy-2[N-propyl-N-(3'-iodo-2'-propenyl)amino]-tetralin maleate were, respectively, 5 and 100 times less effective than quinpirole in inhibiting Ih. 4. Quinpirole failed to reduce Ih when the internal solution contained GDP beta S (500 microM). Internal application GTP gamma S (300 microM) progressively and irreversibly reduced Ih and blocked a further reduction by quinpirole, indicating that the inhibition of Ih by quinpirole involves a G protein. 5. The inhibition of Ih by quinpirole was not affected by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP) or by the protein kinase inhibitor H-7, indicating that a cAMP-mediated second messenger cascade does not participate in the dopamine-mediated inhibition. 6. Ih was not altered when the patch pipette contained a nominally Ca(2+)-free internal solution, but the inhibition of Ih by quinpirole was abolished, suggesting an involvement of Ca(2+) in the quinpirole-induced effect. 7. We conclude that a D2 dopamine receptor modulates Ih through the activation of a G protein and that intracellular Ca2+, but not cAMP, plays a key role in this process. 8. The reduction of Ih by dopamine may reduce the ability of rods to signal time-modulated light stimuli.

1. Using Fura-2, we measured cytosolic free calcium concentrations in rod outer segments of the bullfrog (Rana catesbeiana) under a wide range of steady-state, adapting light intensities. We also measured rod circulating currents under the same adapting conditions. 2. Both the steady-state cytosolic free calcium concentration and the steady-state circulating current were halved by background lights that isomerized approximately 100 rhodopsin molecules per rod per second. A just-measurable reduction in calcium was evoked by a background 1/10 as bright. The steady-state calcium concentration and the steady-state circulating current were proportional to each other over the full range of intensities tested, the brightest of which suppresses the circulating current by 75% and reduces flash sensitivity by about two orders of magnitude relative to the rods' sensitivity in darkness. 3. Additional experiments, in which the Na+:Ca2+,K+ exchanger in the rod outer segment was inactivated while maintaining intracellular calcium at physiological levels, demonstrated that steady-state calcium concentrations in the outer segment are set only by the influx of calcium through the light-sensitive channels and its efflux via the Na+:Ca2+,K+ exchanger. We found no measurable light-induced release of calcium from internal stores and no evidence of any other calcium flux between the cytosol and intracellular compartments. 4. Taken together, these findings lead us to conclude that over the normal operating range of the rod, the selectivity of the cGMP-gated channels for calcium does not change as a result of background illumination. 5. Our data also suggest that a major role of calcium-dependent cGMP synthesis by guanylyl cyclase is to stabilize both the circulating current and the cytosolic free calcium concentration in darkness. This would minimize the dark noise of the rod and thereby increase the reliability with which dim stimuli can be detected in the dark-adapted state.

1. The light responses from one type of ON-OFF amacrine cell were recorded intracellularly in the superfused rabbit retina under various conditions of light adaptation. These recordings were obtained from cells located in a central area. 5-7 mm inferior and directly below the optic nerve head. 2. ON-OFF amacrine cells responded to the initiation and termination of light stimuli with transient depolarizations. Their receptive fields were approximately 0.8-1 mm diam and did not exhibit antagonistic center-and-surround organization. 3. The cells received rod input because they responded to very dim scotopic stimuli. With prolonged dark adaptation, the cells became more sensitive to the initiation than termination of the stimulus, because the ON component of the light response had a lower threshold than the OFF component. 4. The cells continued to respond to test flashes when the retina was adapted to a background illumination of rod-saturating intensity. Thus ON-OFF amacrine cells also receive cone input. Under these photopic conditions, a secondary afterpotential was observed following the OFF component. Its characteristics were different from those of the rod aftereffect reported in other retinal cells of the rabbit because its latency and amplitude changed with increasing stimulus intensity. 5. Intracellular injections of horseradish peroxidase showed that the recordings were obtained from a class of ON-OFF amacrine cells whose wide-field, unistratified dendrites were rigorously confined to the middle of the inner plexiform layer or stratum 3. 6. The conspicuous rod and cone inputs into a class of amacrine cells that are connected neither to rod bipolars nor to All amacrine cells strongly support the idea that in the rabbit the rod pathway uses cone bipolars as interneurons to distribute scotopic signals to ganglion and cone-driven amacrine cells.

1. Single-unit extracellular recordings of optic tract fibers were used to study ganglion cell (GC) response properties of the intact cat eye before and after the intravitreal injection of haloperidol or SCH23390, dopamine-specific antagonists. Nearly all of the dopaminergic cells in the cat retina are amacrine cells (ACs); thus the dopamine antagonists are thought to primarily block the postsynaptic effects of these dopaminergic amacrine cells. All GCs encountered were subjected to a battery of receptive-field (RF) tests, including classification as X or Y, and as ON or OFF. 2. The effects of haloperidol were greatest in the light-adapted OFF-center pathways and especially in the OFF-center Y-cell. Within 30 min of haloperidol injection, both the spontaneous and light-evoked activity of the OFF-center Y-cell fell to zero, but when the same cell was exposed to lower levels of steady-state background illumination (scotopic levels), the response of the cell once again became robust. 3. OFF-Center Y-cells that had partially recovered from the drug effects and OFF-center X-cells recorded when the drug effect was maximal both possessed intensity-response curves that were shifted to the right of normal. 4. Recovery from the drug effects reflect supranormal responses after the initial response reductions and may be due to haloperidol's action on the dopamine autoreceptor. 5. Of the ON-center cells, only the Y-cells showed response alterations; possessing higher spontaneous activities and slightly reduced amplitudes to RF center (RFC) illumination. 6. The effects of SCH23390 paralleled those of haloperidol except that the onset was faster and the duration of the action of SCH23390 was much shorter, and no supranormal responses followed the initial effects. 7. Dark-adaptation functions of OFF-center GCs revealed a normal rod-cone shift; however, SCH23390 eliminated the rod-cone break, and threshold quickly fell to that of the rod mechanism. 8. The dopaminergic neurons of the cat retina appear to play an important role in regulating the activity of retinal OFF-center pathways in the photopically adapted eye, and one of its functions may be to control the relative contributions of the rod and cone systems to the response properties of light-adapted OFF-center GCs. 9. It is argued that dopamine is released in the light and enhances cone pathway activity, perhaps in the outer retina at bipolar and horizontal cells, and suppresses rod pathway activity, perhaps in the inner retina at amacrine cells.

1. The relative effectiveness of adapting lights of different wave-lengths on uncoupling of horizontal cells was measured in dark-adapted mudpuppy retinas. Diffuse blue (470 nm) or red (620 nm) adapting stimuli were adjusted in intensity to be equally effective for rods or for cones. Uncoupling of horizontal cells was measured by intracellular recording of changes in their responses to spot and annulus stimuli. The intensities of the adapting light pairs were varied over 3 log units. The responses of the horizontal cells indicated that both rods and cones were stimulated by the adapting lights. 2. Relatively dim adapting lights did not produce detectable changes in horizontal cell coupling. Brighter adapting lights caused uncoupling of horizontal cells. When the brighter adapting lights were rod matched, the uncoupling effect of the 620-nm light was significantly greater than that of the 470 nm light, indicating that cones contribute to the uncoupling effect. 3. When the adapting lights were cone matched, the effects of the two wavelengths were not significantly different, but this did not rule out a rod contribution because the effective adapting lights probably produced maximal or nearly maximal, and hence equal or nearly equal, responses in rods. 4. The results indicate that cones contribute to the light-evoked uncoupling of horizontal cells in mudpuppy, although a contribution from rods could not be ruled out. Because it was shown previously that light-evoked uncoupling of horizontal cells in mudpuppy is mediated by dopamine, the results also suggest that cones contribute to the light-evoked release of dopamine.

1. Two rod-driven electroretinogram (ERG) components were recorded to monitor scotopic retinal signals during experimental manipulation of dopamine and gamma-aminobutyric acid (GABA) in normal cat eyes and in eyes pretreated with 6-hydroxydopamine (6-OHDA). The scotopic threshold response (STR) was elicited near absolute threshold to monitor signals traversing the rod pathway near quantal threshold; scotopic PII, which normally begins approximately 2 log units higher, was also monitored. Responses were evaluated by V-log I curves and criterion amplitudes after intravitreal drug injections into intact eyes in vivo. The depletion of dopaminergic cells by pretreating with 6-OHDA was confirmed histologically by immunocytochemical methods. 2. Dopamine abolished the STR and markedly decreased PII in the normal eye. Both 6-OHDA pretreatment and application of the dopamine antagonist, haloperidol, increased the STR amplitudes, but only for stimuli beginning 2 log units above threshold; PII amplitude also was increased. However, neither 6-OHDA pretreatment nor haloperidol affected the STR near absolute threshold. 3. Both GABA and bicuculline suppressed the STR in the normal eye. However, when applied to eyes pretreated with 6-OHDA or concurrent with haloperidol, bicuculline enhanced the STR. GABA enhanced the PII amplitude in the normal eye but had no effect in eyes pretreated with 6-OHDA or in the presence of haloperidol. 4. These results suggest that 1) dopaminergic activity modulates signals in the rod pathway at higher stimulus intensities but not near absolute threshold and 2) GABA can affect the scotopic PII component by acting through dopaminergic cells in the dark-adapted retina.