TRPV4 affects visual signals in photoreceptors and rod bipolar cells.

Introduction: Mechanical sensitive channels expressed in mammalian retinas are effectors of elevated pressure stresses, but it is unclear how their activation affects visual function in pressure-related retinal disorders. Methods: This study investigated the role of the transient potential channel vanilloid TRPV4 in photoreceptors and rod bipolar cells (RBCs) with immunohistochemistry, confocal microscopy, electroretinography (ERG), and patch-clamp techniques. Results: TRPV4 immunoreactivity (IR) was found in the outer segments of photoreceptors, dendrites and somas of PKCα-positive RBCs and other BCs, plexiform layers, and retinal ganglion cells (RGCs) in wild-type mice. TRPV4-IR was largely diminished in the retinas of homozygous TRPV4 transgenic mice. Genetically suppressing TRPV4 expression moderately but significantly enhanced the amplitude of ERG a- and b-waves evoked by scotopic and mesopic lights (0.55 to 200 Rh*rod-1 s-1) and photopic lights (105-106 Rh*rod-1 s-1) compared to wild-type mice in fully dark-adapted conditions. The implicit time evoked by dim lights (0.55 to 200 Rh*rod-1 s-1) was significantly decreased for b-waves and elongated for a-waves in the transgenic mice. ERG b-wave evoked by dim lights is primarily mediated by RBCs, and under voltage-clamp conditions, the latency of the light-evoked cation current in RBCs of the transgenic mice was significantly shorter compared to wild-type mice. About 10% of the transgenic mice had one eye undeveloped, and the percentage was significantly higher than in wild-type mice. Conclusions: The data indicates that TRPV4 involves ocular development and is expressed and active in outer retinal neurons, and interventions of TRPV4 can variably affect visual signals in rods, cones, RBCs, and cone ON BCs.

The Variety of Mechanosensitive Ion Channels in Retinal Neurons.

Alterations in intraocular and external pressure critically involve the pathogenesis of glaucoma, traumatic retinal injury (TRI), and other retinal disorders, and retinal neurons have been reported to express multiple mechanical-sensitive channels (MSCs) in recent decades. However, the role of MSCs in visual functions and pressure-related retinal conditions has been unclear. This review will focus on the variety and functional significance of the MSCs permeable to K+, Na+, and Ca2+, primarily including the big potassium channel (BK); the two-pore domain potassium channels TRAAK and TREK; Piezo; the epithelial sodium channel (ENaC); and the transient receptor potential channels vanilloid TRPV1, TRPV2, and TRPV4 in retinal photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Most MSCs do not directly mediate visual signals in vertebrate retinas. On the other hand, some studies have shown that MSCs can open in physiological conditions and regulate the activities of retinal neurons. While these data reasonably predict the crossing of visual and mechanical signals, how retinal light pathways deal with endogenous and exogenous mechanical stimulation is uncertain.

Linear and Nonlinear Behaviors of the Photoreceptor Coupled Network.

Photoreceptors are electrically coupled to one another, and the spatiotemporal properties of electrical synapses in a two-dimensional retinal network are still not well studied, because of the limitation of the single electrode or pair recording techniques which do not allow simultaneously measuring responses of multiple photoreceptors at various locations in the retina. A multiple electrode recording system is needed. In this study, we investigate the network properties of the two-dimensional rod coupled array of the salamander retina (both sexes were used) by using the newly available multiple patch electrode system that allows simultaneous recordings from up to eight cells and to determine the electrical connectivity among multiple rods. We found direct evidence that voltage signal spread in the rod-rod coupling network in the absence of I h (mediated by HCN channels) is passive and follows the linear cable equation. Under physiological conditions, I h shapes the network signal by progressively shortening the response time-to-peak of distant rods, compensating the time loss of signal traveling from distant rods to bipolar cell somas and facilitating synchronization of rod output signals. Under voltage-clamp conditions, current flow within the coupled rods follows Ohm's law, supporting the idea that nonlinear behaviors of the rod network are dependent on membrane voltage. Rod-rod coupling is largely symmetrical in the 2D array, and voltage-clamp blocking the next neighboring rod largely suppresses rod signal spread into the second neighboring rod, suggesting that indirect coupling pathways play a minor role in rod-rod coupling.

Light responses and amacrine cell modulation of morphologically-identified retinal ganglion cells in the mouse retina.

By analyzing light-evoked spike responses, cation currents (ΔIC) and chloride currents (ΔICl) of over 100 morphologically-identified retinal ganglion cells (GCs) in dark-adapted mouse retina, we found there are at least 14 functionally- and morphologically-distinct types of RGCs. These cells can be divided into 5 groups based on their patterns of spike response to whole field light steps (SRWFLS), a GC identification scheme commonly used in studies with extracellular recording techniques. We also found that all GCs in the mouse retina express strychnine-sensitive glycine receptors, and receive light-elicited chloride current (ΔICl) accompanied by a conductance increase from narrow-field, glycinergic amacrine cells. As the dark membrane potential of RGC are near the chloride-equilibrium potential, mouse GCs' spike responses are mediated primarily by bipolar cells inputs, and modulated by "shunting inhibition" from narrow-field amacrine cells. Analysis of strychnine actions on light-evoked cation current ΔIC (bipolar cell inputs) in GCs suggests that narrow-field amacrine cells modulate GCs by sending ON-OFF crossover feedback signals to presynaptic bipolar cell axon terminals via sign-inverting glycinergic synapses, and the feedback signals are synergistic to the bipolar cell light responses. Therefore narrow-field amacrine cells enhance light-evoked bipolar cell inputs to GCs by presynaptic "synergistic addition", besides the abovementioned postsynaptic "shunting inhibition" in GCs.

Dual-Cell Patch-Clamp Recording Revealed a Mechanism for a Ribbon Synapse to Process Both Digital and Analog Inputs and Outputs.

A chemical synapse is either an action potential (AP) synapse or a graded potential (GP) synapse but not both. This study investigated how signals passed the glutamatergic synapse between the rod photoreceptor and its postsynaptic hyperpolarizing bipolar cells (HBCs) and light responses of retinal neurons with dual-cell and single-cell patch-clamp recording techniques. The results showed that scotopic lights evoked GPs in rods, whose depolarizing Phase 3 associated with the light offset also evoked APs of a duration of 241.8 ms and a slope of 4.5 mV/ms. The depolarization speed of Phase 3 (Speed) was 0.0001-0.0111 mV/ms and 0.103-0.469 mV/ms for rods and cones, respectively. On pairs of recorded rods and HBCs, only the depolarizing limbs of square waves applied to rods evoked clear currents in HBCs which reversed at -6.1 mV, indicating cation currents. We further used stimuli that simulated the rod light response to stimulate rods and recorded the rod-evoked excitatory current (rdEPSC) in HBCs. The normalized amplitude (R/Rmax), delay, and rising slope of rdEPSCs were differentially exponentially correlated with the Speed (all p < 0.001). For the Speed < 0.1 mV/ms, R/Rmax grew while the delay and duration reduced slowly; for the Speed between 0.1 and 0.4 mV/ms, R/Rmax grew fast while the delay and duration dramatically decreased; for the Speed > 0.4 mV/ms, R/Rmax reached the plateau, while the delay and duration approached the minimum, resembling digital signals. The rdEPSC peak was left-shifted and much faster than currents in rods. The scotopic-light-offset-associated major and minor cation currents in retinal ganglion cells (RGCs), the gigantic excitatory transient currents (GTECs) in HBCs, and APs and Phase 3 in rods showed comparable light-intensity-related locations. The data demonstrate that the rod-HBC synapse is a perfect synapse that can differentially decode and code analog and digital signals to process enormously varied rod and coupled-cone inputs.

Ocular Pressure-Volume Relationship and Ganglion Cell Death in Glaucoma.

We studied how GC death in glaucoma related to the intraocular pressure (IOP), eyeball volume (VS) and elasticity (volumetric KS and tensile ES), and eyeball volume-pressure relation. Glaucomatous GC loss was studied in DBA/2J (D2) mice with wild-type mice as controls. GCs were retrogradely identified and observed with a confocal microscope. The elasticity calculation was also done on published data from patients treated by a gas bubble injection in the vitreous cavity. The GC population in D2 mice (1.5- to 14-month-old) was negatively correlated with following factors: VS (p = 0.0003), age (p = 0.0026) and IOP (but p = 0.0966). As indicated by average values, adult D2 mice (≥6 months) suffered significant GC loss, low KS and ES, and universal expansion of VS with normal IOP. KS and ES in the patients were also lower upon prolonged eyeball expansion compared to acute expansion. Based on the results and presumptions of a closed and continuous eyeball space (thereby ΔVS ≈ ΔVW, ΔVW-the change in the aqueous humor amount), we deduced equations on the ocular volume-pressure relationship: ΔIOP = KS*ΔVW/VS or ΔIOP = (2/3)*[1/(1-ν)]*(H/R)*ES*ΔVW/VS (ν, Poisson's ratio taken as 0.5; R, the curvature radius; and H, the shell thickness). Under normal atmospheric pressure, IOP of 10~50 mmHg contributed only 1.2~6.6% of the pressure opposing the retina and eyeball shell. We conclude: 1) A disturbance of ocular volume-pressure homeostasis, mediated primarily by low KS and ES, expanded VS, and large ΔVW, is correlated with GC death in glaucoma and 2) D2 mice with GC loss and normal IOP may serve as animal models for human normal-tension glaucoma.

Generators of Pressure-Evoked Currents in Vertebrate Outer Retinal Neurons.

(1) Background: High-tension glaucoma damages the peripheral vision dominated by rods. How mechanosensitive channels (MSCs) in the outer retina mediate pressure responses is unclear. (2) Methods: Immunocytochemistry, patch clamp, and channel fluorescence were used to study MSCs in salamander photoreceptors. (3) Results: Immunoreactivity of transient receptor potential channel vanilloid 4 (TRPV4) was revealed in the outer plexiform layer, K+ channel TRAAK in the photoreceptor outer segment (OS), and TRPV2 in some rod OS disks. Pressure on the rod inner segment evoked sustained currents of three components: (A) the inward current at <-50 mV (Ipi), sensitive to Co2+; (B) leak outward current at ≥-80 mV (Ipo), sensitive to intracellular Cs+ and ruthenium red; and (C) cation current reversed at ~10 mV (Ipc). Hypotonicity induced slow currents like Ipc. Environmental pressure and light increased the FM 1-43-identified open MSCs in the OS membrane, while pressure on the OS with internal Cs+ closed a Ca2+-dependent current reversed at ~0 mV. Rod photocurrents were thermosensitive and affected by MSC blockers. (4) Conclusions: Rods possess depolarizing (TRPV) and hyperpolarizing (K+) MSCs, which mediate mutually compensating currents between -50 mV and 10 mV, serve as an electrical cushion to minimize the impact of ocular mechanical stress.

Roles of the ocular pressure, pressure-sensitive ion channel, and elasticity in pressure-induced retinal diseases.

The intraocular pressure inside the human eye maintains 10-21 mmHg above the atmospheric pressure. Elevation of intraocular pressure is highly correlated with the retinopathy in glaucoma, and changes in the exterior pressure during mountain hiking, air traveling, and diving may also induce vision decline and retinopathy. The pathophysiological mechanism of these pressure-induced retinal disorders has not been completely clear. Retinal neurons express pressure-sensitive channels intrinsically sensitive to pressure and membrane stretch, such as the transient receptor potential channel (TRP) family permeable to Ca2+ and Na+ and the two-pore domain K channel family. Recent data have shown that pressure excites the primate retinal bipolar cell by opening TRP vanilloid 4 to mediate transient depolarizing currents, and TRP vanilloid 4 agonists enhance the membrane excitability of primate retinal ganglion cells. The eyeball wall is constructed primarily by the sclera and cornea of low elasticity, and the flow rate of the aqueous humor and intraocular pressure both fluctuate, but the mathematical relationship between the ocular elasticity, aqueous humor volume, and intraocular pressure has not been established. This review will briefly review recent literature on the pressure-related retinal pathophysiology in glaucoma and other pressure-induced retinal disorders, the elasticity of ocular tissues, and pressure-sensitive cation channels in retinal neurons. Emerging data support the global volume and the elasticity and thickness of the sclera and cornea as variables to affect the intraocular pressure level like the volume of the aqueous humor. Recent results also suggest some potential routes for TRPs to mediate retinal ganglion cell dysfunction: TRP opening upon intraocular pressure elevation and membrane stretch, enhancing glutamate release from bipolar cells, increasing intracellular Na+, Ca2+ concentration in retinal ganglion cells and extracellular glutamate concentration, inactivating voltage-gated Na+ channels, and causing excitotoxicity and dysfunction of retinal ganglion cells. Further studies on these routes likely identify novel targets and therapeutic strategies for the treatment of pressure-induced retinal disorders.

The expression and function of TRPV4 channels in primate retinal ganglion cells and bipolar cells.

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.

Cone synapses in mammalian retinal rod bipolar cells.

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.

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

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

Elevated intraocular pressure decreases response sensitivity of inner retinal neurons in experimental glaucoma mice.

Glaucoma is the second leading cause of blindness in the United States and the world, characterized by progressive degeneration of the optic nerve and retinal ganglion cells (RGCs). Glaucoma patients exhibit an early diffuse loss of retinal sensitivity followed by focal loss of RGCs in sectored patterns. Recent evidence has suggested that this early sensitivity loss may be associated with dysfunctions in the inner retina, but detailed cellular and synaptic mechanisms underlying such sensitivity changes are largely unknown. In this study, we use whole-cell voltage-clamp techniques to analyze light responses of individual bipolar cells (BCs), AII amacrine cells (AIIACs), and ON and sustained OFF alpha-ganglion cells (ONαGCs and sOFFαGCs) in dark-adapted mouse retinas with elevated intraocular pressure (IOP). We present evidence showing that elevated IOP suppresses the rod ON BC inputs to AIIACs, resulting in less sensitive AIIACs, which alter AIIAC inputs to ONαGCs via the AIIAC→cone ON BC→ONαGC pathway, resulting in lower ONαGC sensitivity. The altered AIIAC response also reduces sOFFαGC sensitivity via the AIIAC→sOFFαGC chemical synapses. These sensitivity decreases in αGCs and AIIACs were found in mice with elevated IOP for 3-7 wk, a stage when little RGC or optic nerve degeneration was observed. Our finding that elevated IOP alters neuronal function in the inner retina before irreversible structural damage occurs provides useful information for developing new diagnostic tools and treatments for glaucoma in human patients.

Sign-preserving and sign-inverting synaptic interactions between rod and cone photoreceptors in the dark-adapted retina.

We show that various types of rods and cones in the dark-adapted salamander retina are electrically coupled with linear and symmetrical junctional conductances G(j) (40-223 pS) and a rank order: Rod(C)-large single cone, rod-large single cone, rod-small single cone, rod-accessory double cone and rod-principal double cone. By systematically comparing the transjunctional current-voltage (I(j)-V(j)) relations and average G(j) values of the five types of rod-cone pairs recorded at day and night times, our results suggest that the differences in G(j) values among various types of rod-cone pairs are not caused by circadian differences, and the circadian-dependent changes in rod-cone coupling observed in the fish and rodent retinas are not present in the tiger salamander. In addition to rod-cone coupling, there is a sign-inverting, unidirectional rod→cone current I(RC), and the I(RC)-V(Cone) relations are linear, with a reversal potential near the chloride reversal potential E(Cl). I(RC) can be observed in rods and cones separated by at least 260 µm, and its waveform resembles that of the rod-elicited horizontal cell (HC) response I(HC). A glutamate transporter-associated chloride channel blocker TBOA suppresses I(RC) but not I(HC). These results suggest that I(RC) is largely mediated by HCs via a sign-inverting feedback chemical synapse associated with a chloride channel. I(RC) significantly reduced rod→cone coupling in the frequency range below 15 Hz, allowing better separation of rod and cone signals in the dark-adapted retina.

Survey on amacrine cells coupling to retrograde-identified ganglion cells in the mouse retina.

PURPOSE: Retinal amacrine cells (ACs) may make inhibitory chemical synapses and potentially excitatory gap junctions on ganglion cells (GCs). The total number and subtypes of ACs coupled to the entire GC population were investigated in wild-type and three lines of transgenic mice. METHODS: GCs and GC-coupled ACs were identified by the previously established LY-NB (Lucifer yellow-Neurobiotin) retrograde double-labeling technique, in conjunction with specific antibodies and confocal microscopy. RESULTS: GC-coupled ACs (NB-positive and LY-negative) comprised nearly 11% of displaced ACs and 4% of conventional ACs in wild-type mice, and were 9% and 4% of displaced ACs in Cx45(-/-) and Cx36/45(-/-) mice, respectively. Their somas were small in Cx36/45(-/-) mice, but variable in other strains. They were mostly γ-aminobutyric acid (GABA)-immunoreactive (IR) and located in the GC layer. They comprised only a small portion in the AC subpopulations, including GABA-IR, glycine-IR, calretinin-IR, 5-HT-accumulating, and ON-type choline acetyltransferase (ChAT) ACs in wild-type and ChAT transgenic mice (ChAT- tdTomato). In the distal 80% of the inner plexiform layer (IPL), dense GC dendrites coexisted with rich glycine-IR and GABA-IR. In the inner 20% of the IPL, sparse GC dendrites presented with a major GABA band and sparse glycine-IR. CONCLUSIONS: Various subtypes of ACs may couple to GCs. ACs of the same immunoreactivity may either couple or not couple to GCs. Cx36 and Cx45 dominate GC-AC coupling except for small ACs. The overall potency of GC-AC coupling is moderate, especially in the proximal 20% of the IPL, where inhibitory chemical signals are dominated by GABA ACs.

Elevated intraocular pressure causes inner retinal dysfunction before cell loss in a mouse model of experimental glaucoma.

PURPOSE: We assessed the relationship among intraocular pressure (IOP), histology, and retinal function changes in a mouse model of induced, chronic, mild ocular hypertension. METHODS: IOP was elevated experimentally via anterior chamber injection of polystyrene beads and measured twice weekly with a rebound tonometer. Histology was assessed with a combination of neurobiotin (NB) retrograde labeling of retinal ganglion cells (RGCs) and TO-PRO3 staining. Retinal function was assessed with serial dark-adapted electroretinograms (ERGs) optimized for detection of the a-wave, b-wave, and positive and negative scotopic threshold responses (pSTR, nSTR). Comparisons between bead-injected and saline-injected (control) eyes were conducted. RESULTS: IOP remained elevated for at least 3 months following a single injection of polystyrene beads. Elevated IOP resulted in a mild, progressive reduction of RGCs, and a mild increase in axial length at 6 and 12 weeks after bead injection. The raw b-wave amplitude was increased shortly after IOP elevation, but the raw a-wave, pSTR, and nSTR amplitudes were unchanged. pSTR and nSTR amplitudes were normalized to the increased b-wave. With this normalization, the pSTR amplitude was decreased shortly after IOP elevation. CONCLUSIONS: Polystyrene bead injection results in a mild, chronic elevation of IOP that recapitulates several critical aspects of human ocular hypertension and glaucoma, and results in early changes in retinal electrical function that precede histologic changes. It is possible that glaucoma associated with elevated IOP involves the early disruption of a complex combination of retinal synapses.

Ionotropic glutamate receptors mediate OFF responses in light-adapted ON bipolar cells.

Previous studies have suggested that photoreceptor synaptic inputs to depolarizing bipolar cells (DBCs or ON bipolar cells) are mediated by mGluR6 receptors and those to hyperpolarizing bipolar cells (HBCs or OFF bipolar cells) are mediated by AMPA/kainate receptors. Here we show that in addition to mGluR6 receptors which mediate the sign-inverting, depolarizing light responses, subpopulations of cone-dominated and rod/cone mixed DBCs use GluR4 AMPA receptors to generate a transient sign-preserving OFF response under light adapted conditions. These AMPA receptors are located at the basal junctions postsynaptic to rods and they are silent under dark-adapted conditions, as tonic glutamate release in darkness desensitizes these receptors. Light adaptation enhances rod-cone coupling and thus allows cone photocurrents with an abrupt OFF depolarization to enter the rods. The abrupt rod depolarization triggers glutamate activation of unoccupied AMPA receptors, resulting in a transient OFF response in DBCs. It has been widely accepted that the DNQX-sensitive, OFF transient responses in retinal amacrine cells and ganglion cells are mediated exclusively by HBCs. Our results suggests that this view needs revision as AMPA receptors in subpopulations of DBCs are likely to significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons.

Rod, M-cone and M/S-cone inputs to hyperpolarizing bipolar cells in the mouse retina.

Bipolar cells are the central neurons of the retina that convey visual signals from rod and cone photoreceptors in the outer retina to higher-order neurons in the inner retina and the brain. Early anatomical studies have suggested that there are four types of cone hyperpolarizing (OFF) bipolar cells (HBCs) in the mouse retina, but no light responses have been systematically examined. By analysing light-evoked cation and chloride currents (I(C) and I(Cl)) from over 50 morphologically identified HBCs in the dark-adapted wildtype and connexin36 knockout (Cx36(-/-)) mouse retinas, we identified three types of HBCs, each with distinct light responses and morphological characteristics. The HBC(R/MC)s with axon terminals ramifying between 0% and 30% of the inner plexiform layer (IPL) receive mixed inputs from rods and M-cones, the HBC(MC)s with axon terminals ramifying between 10% and 50% of the IPL receive inputs primarily from M-cones, and the HBC(M/SC)s with axon terminals ramifying between 25% and 50% of IPL receive inputs primarily from cones with mixed M- and S-cone pigments. Moreover, we found that HBC(R/MC)s in the Cx36(-/-) mice exhibit light responses very similar to the wildtype HBC(R/MC)s, suggesting that the mixed rod-cone inputs are not mediated by connexin36-dependent rod-cone coupling, but rather by direct synaptic contacts from rods and M-cones. This study constitutes the first systematic investigation that correlates light response characteristics and axonal morphology of HBCs in dark-adapted mouse retina, and contributes to recently emerging evidence that revises the traditional view that mammalian HBCs only contact cone photoreceptors.

Physiological characterization and functional heterogeneity of narrow-field mammalian amacrine cells.

Light-evoked responses of 106 morphologically identified narrow-field amacrine cells (ACs) were studied in dark-adapted mouse retinal slices. Forty-five cells exhibit AIIAC morphology, 55% of which show characteristic AIIAC physiological properties (AIIAC1s) and the remaining 45% display different physiological responses, suggesting that AIIACs are functionally heterogeneous. Moreover, we found that 42 cells exhibit morphology that resembles the seven morphological types of glycine-positive ACs (GlyAC1-7) reported in the rat retina, and for the first time assigned light response and function properties to these morphological types of glycinergic ACs in the mouse retina. In addition, five narrow-field ACs exhibited morphology resembling that of the GlyAC5 or GlyAC7 but with different physiological responses (GlyAC5(#) and GlyAC7(#)). Therefore, the eight morphological types of narrow-field ACs exhibit 12 classes of physiological responses. Furthermore, we found ACs whose physiological responses were indistinguishable from those of GlyAC3 or GlyAC4s but with different morphology (GlyAC3* or GlyAC4*). These observations suggest that although the majority of narrow-field mammalian ACs forms discrete functional groups that correlate with their morphology, a significant number of these cells with similar morphology do not display the same light responses, and some with similar light responses do not exhibit the same morphology.

Morphology and immunoreactivity of retrogradely double-labeled ganglion cells in the mouse retina.

PURPOSE: To examine the specificity and reliability of a retrograde double-labeling technique that was recently established for identification of retinal ganglion cells (GCs) and to characterize the morphology of displaced (d)GCs (dGs). METHODS: A mixture of the gap-junction-impermeable dye Lucifer yellow (LY) and the permeable dye neurobiotin (NB) was applied to the optic nerve stump for retrograde labeling of GCs and the cells coupled with them. A confocal microscope was adopted for morphologic observation. RESULTS: GCs were identified by LY labeling, and they were all clearly labeled by NB. Cells coupled to GCs contained a weak NB signal but no LY. LY and NB revealed axon bundles, somas and dendrites of GCs. The retrogradely identified GCs numbered approximately 50,000 per retina, and they constituted 44% of the total neurons in the ganglion cell layer (GCL). Somas of retrogradely identified dGs were usually negative for glycine, ChAT (choline acetyltransferase), bNOS (brain-type nitric oxidase), GAD (glutamate decarboxylase), and glial markers, and occasionally, they were weakly GABA-positive. dGs averaged 760 per retina and composed 1.7% of total GCs. Sixteen morphologic subtypes of dGs were encountered, three of which were distinct from known GCs. dGs sent dendrites to either sublaminas of the IPL, mostly sublamina a. CONCLUSIONS: The retrograde labeling is reliable for identification of GCs. dGs participate in ON and OFF light pathways but favor the OFF pathway. ChAT, bNOS, glycine, and GAD remain reliable AC markers in the GCL. GCs may couple to GABAergic ACs, and the gap junctions likely pass NB and GABA.

Light responses and morphology of bNOS-immunoreactive neurons in the mouse retina.

Nitric oxide (NO), produced by NO synthase (NOS), modulates the function of all retinal neurons and ocular blood vessels and participates in the pathogenesis of ocular diseases. To further understand the regulation of ocular NO release, we systematically studied the morphology, topography, and light responses of NOS-containing amacrine cells (NOACs) in dark-adapted mouse retina. Immunohistological staining for neuronal NOS (bNOS), combined with retrograde labeling of ganglion cells (GCs) with Neurobiotin (NB, a gap junction permeable dye) and Lucifer yellow (LY, a less permeable dye), was used to identify NOACs. The light responses of ACs were recorded under whole-cell voltage clamp conditions and cell morphology was examined with a confocal microscope. We found that in dark-adapted conditions bNOS-immunoreactivity (IR) was present primarily in the inner nuclear layer and the ganglion cell layer. bNOS-IR somas were negative for LY, thus they were identified as ACs; nearly 6% of the cells were labeled by NB but not by LY, indicating that they were dye-coupled with GCs. Three morphological subtypes of NOACs (NI, NII, and displaced) were identified. The cell density, intercellular distance, and the distribution of NOACs were studied in whole retinas. Light evoked depolarizing highly sensitive ON-OFF responses in NI cells and less sensitive OFF responses in NII cells. Frequent (1-2 Hz) or abrupt change of light intensity evoked larger peak responses. The possibility for light to modify NO release from NOACs is discussed.