Samuel M. Wu

A quantitative analysis of interactions between photoreceptors in the salamander (Ambystoma) retina

A quantitative description of the electrical properties of the photoreceptor layer in the salamander retina was obtained from earlier data on the characteristics of isolated rods and cones and on rod‐rod coupling, and from new data on rod‐cone and cone‐cone coupling and on the rod photocurrent. Injecting ‐1 nA current into a rod elicits hyperpolarizations of about 20 mV in an adjacent rod and 4 mV in an adjacent cone. Responses of more distant receptors are smaller. Injecting ‐1 nA into a cone elicits hyperpolarizations of about 4 mV in an adjacent rod and 0.4 mV in a nearby cone. Depolarizing current evokes smaller responses. Assuming, in agreement with anatomical evidence, that each rod is electrically coupled to four rods and to four cones around it, and that there is no direct electrical coupling between cones, we found these results could be predicted from the properties of isolated rods and cones if adjacent rods are coupled by a resistance of 300 M omega and adjacent rods and cones are coupled by a resistance of 5000 M omega. The small cone‐cone coupling seen is due to coupling via intervening rods. The two halves of double cones are not electrically coupled. The spectral sensitivity of both halves is a maximum around 620 nm wave‐length. The rod photocurrent has been characterized by voltage‐clamping rods isolated from the retina. In agreement with Bader, MacLeish & Schwartz (1979) we found the time course of the photocurrent to be approximately independent of voltage between ‐35 and ‐85 mV. The voltage responses of rods, single cones and double cones isolated from the retina obey the principle of univariance. Responses of receptors in the retina do not obey univariance. The main deviations from univariance observed can be explained if adjacent rods and cones are coupled by a resistance of 5000 M omega. Our data demonstrate that rod‐cone coupling is relatively weak. We simplified our description of the photoreceptor network, by omitting cones, to investigate the spatiotemporal processing that the rod network is capable of. Computer simulations predict, as is found experimentally, that the rod voltage response to a large spot of bright light should show a much more pronounced initial transient hyperpolarization than the response to a small spot of light of the same intensity. This difference is produced by the combination of electrical coupling of the rods with the existence of a voltage‐gated current, IA, in the rod membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Melatonin enhances horizontal cell sensitivity in salamander retina

Intracellular electrophysiological recording techniques were utilized to investigate the possible function of retinal melatonin in the larval tiger salamander. Endogenous retinal melatonin was present and appeared to bind a membrane-enriched fraction of the salamander retina, as determined by radioimmunoassay and receptor binding studies. Melatonin added through the perfusion bath to flat-mounted retinas resulted in a horizontal cell (HC) hyperpolarization of 10-20 mV. Additionally, the amplitude of HC responses to short test flashes increased in the presence of melatonin. Voltage-intensity plots revealed that application of 500 microM of melatonin caused an increase of the HC light sensitivity and this effect was reversible. These results suggest that melatonin synthesized and released during the dark period of the diurnal cycle may alter the sensitivity of second-order neurons at a time of day when photopic input is at its lowest level.

Amino acid neurotransmitters in the retina : a functional overview

Physiological and pharmacological mechanisms of glutamatergic, GABAergic and glycinergic synapses in the tiger salamander retina were studied. We used immunocytochemical and autoradiographic methods to study localizations of these neurotransmitters and their uptake transporters; and electrophysiological methods (intracellular, extracellular and whole cell patch electrode recordings) to study the light responses, miniature postsynaptic currents and neurotransmitter-induced postsynaptic currents in various retinal neurons. Our results are consistent with the following scheme: Glutamate is used by the photoreceptor and bipolar cell output synapses and the release of glutamate is largely mediated by calcium-dependent vesicular processes. The postsynaptic glutamate receptors in DBCs are L-AP4 receptors, in HBCs, HCs and ganglion cells are the kainate/AMPA and NMDA receptors. Subpopulations of HCs make GABAergic synapses on cones and gate chloride condunctance through GABAA receptors. GABAergic HCs do not make feedforward synapses on bipolar cell dendrites and the neurotransmitter identity of the HCs making feedforward synapses is unknown. Subpopulations of amacrine cells make GABAergic synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and GABA gates chloride conductances in theses cells. Glycinergic amacrine cells make synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and glycine opens postsynaptic chloride channels. Glycinergic interplexiform cells make synapses on bipolar cells in the outer retina and glycine released from these cells open chloride channels in bipolar cell dendrites.

Light-evoked current responses in rod bipolar cells, cone depolarizing bipolar cells and AII amacrine cells in dark-adapted mouse retina.

Light‐evoked excitatory cation current (ΔIC) and inhibitory chloride current (ΔICl) of rod and cone depolarizing bipolar cells (DBCRs and DBCCs) and AII amacrine cells (AIIACs) in dark‐adapted mouse retinal slices were studied by whole‐cell voltage‐clamp recording techniques, and the cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. ΔIC of all DBCRs exhibited similar high sensitivity to 500 nm light, but two patterns of ΔICl were observed in DBCRs with slightly different axon morphology. At least two types of DBCCs were identified: one with axon terminals ramified in 70–85% of the depth of the inner plexiform layer (IPL) and DBCR‐like ΔIC sensitivity, whereas the other with axon terminals ramified in 55–75% of IPL depth and much lower ΔIC sensitivity. The relative rod/cone inputs to DBCs and AIIACs were analysed by comparing the ΔIC and ΔICl thresholds and dynamic ranges with the corresponding values of rods and cones. On average, the sensitivity of a DBCR to the 500 nm light is about 20 times higher than that of a rod. The sensitivity of an AIIAC is more than 1000 times higher than that of a rod, suggesting that AIIAC responses are pooled through a coupled network of about 40 AIIACs. Interactions of rod and cone signals in dark‐adapted mouse retina appear asymmetrical: rod signals spread into the cone system more efficiently than cone signals into the rod system. The mouse synaptic circuitry allows small rod signals to be highly amplified, and effectively transmitted to the cone system via rod–cone and AIIAC–DBCC coupling.

Effects of gamma-aminobutyric acid on cones and bipolar cells of the tiger salamander retina.

The gamma-aminobutyric acid (GABA) system in the tiger salamander retina was studied using autoradiographic and electrophysiological techniques. A high-affinity uptake mechanism for GABA has been localized in about 60% of the horizontal cells and about 30% of the amacrine cells. Effects of exogeneously applied GABA on the membrane conductance of cones and hyperpolarizing bipolar cells (HBC) were examined using the two electrode current-clamp technique in the living retinal slices. In both cell types, 1 mM of GABA caused a conductance increase. In perfused eyecups, 2 mM of GABA selectivity abolished the surround response of the HBC and left the center response unchanged. These results are consistent with the notion that a population of horizontal cells and a population of amacrine cells in the salamander retina may use GABA as their neurotransmitter.

Retinal ganglion cell dysfunction induced by hypoxia and glutamate: potential neuroprotective effects of beta-blockers.

The objective of this study was to examine the effects of hypoxia, glutamate, and beta-blockers on the electrical activities of retinal ganglion cells. Single-unit extracellular and whole-cell voltage clamp recording techniques were used to record electrical activities from ganglion cells in the tiger salamander retina. This was performed under physiologic conditions, hypoxia, or elevated exogenous or endogenous glutamate levels. Light-evoked spike activities, glutamate-induced currents, and voltage-gated sodium and calcium currents were measured in the presence of the beta-1 selective antagonist betaxolol or the nonselective antagonist timolol. Hypoxia resulted in suppressing or blocking the OFF responses in the majority of ON-OFF ganglion cells tested, whereas the ON responses were only slightly affected. The presence of increased glutamate had similar findings and demonstrated an increase in the spontaneous firing rate of retinal ganglion cells. Betaxolol (2-50 microM) reduced the rate of spontaneous firing of retinal ganglion cells induced by glutamate. At 2 to 50 microM, betaxolol reversibly reduced the voltage-gated sodium currents and calcium currents in retinal ganglion cells. Timolol (up to 100 microM) did not demonstrate any detectable action on these currents. The physiologic responses of retinal ganglion cells to hypoxia or elevated glutamate levels in this animal model appear to be very similar. Although short-term exposure to hypoxia and glutamate used in this study exerts reversible actions on ganglion cells and does not induce permanent cell damage, such initial physiologic actions are likely to be precursors of permanent cell damage. Thus, hypoxia and elevated glutamate levels in the retina may represent a final pathway in diseases affecting retinal ganglion cells, such as glaucoma. Similar damage could result from different factors, such as decreased perfusion-induced ischemia or anomalous neuronal processing of glutamate. Betaxolol exerts its primary neuronal actions on retinal ganglion cells. It reversibly blocked voltage-gated calcium current and reduced the spontaneous firing rate by suppressing glutamate-gated currents and sodium currents in ganglion cells. These actions may protect ganglion cells from damage caused by ischemia or elevated glutamate levels.

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

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

Gene therapy prevents photoreceptor death and preserves retinal function in a Bardet-Biedl syndrome mouse model

Patients with Bardet-Biedl syndrome (BBS) experience severe retinal degeneration as a result of impaired photoreceptor transport processes that are not yet fully understood. To date, there is no effective treatment for BBS-associated retinal degeneration, and blindness is imminent by the second decade of life. Here we report the development of an adeno-associated viral (AAV) vector that rescues rhodopsin mislocalization, maintains nearly normal-appearing rod outer segments, and prevents photoreceptor death in the Bbs4-null mouse model. Analysis of the electroretinogram a-wave indicates that rescued rod cells are functionally indistinguishable from wild-type rods. These results demonstrate that gene therapy can prevent retinal degeneration in a mammalian BBS model.

Direct rod input to cone BCs and direct cone input to rod BCs challenge the traditional view of mammalian BC circuitry

Bipolar cells are the central neurons of the retina that transmit visual signals from rod and cone photoreceptors to third-order neurons in the inner retina and the brain. A dogma set forth by early anatomical studies is that bipolar cells in mammalian retinas receive segregated rod/cone synaptic inputs (either from rods or from cones), and here, we present evidence that challenges this traditional view. By analyzing light-evoked cation currents from morphologically identified depolarizing bipolar cells (DBCs) in the wild-type and three pathway-specific knockout mice (rod transducin knockout [Trα−/−], connexin36 knockout [Cx36−/−], and transcription factor beta4 knockout [Bhlhb4−/−]), we show that a subpopulation of rod DBCs (DBCR2s) receives substantial input directly from cones and a subpopulation of cone DBCs (DBCC1s) receives substantial input directly from rods. These results provide evidence of the existence of functional rod-DBCC and cone-DBCR synaptic pathways in the mouse retina as well as the previously proposed rod hyperpolarizing bipolar-cells pathway. This is grounds for revising the mammalian rod/cone bipolar cell dogma.

The retinoid cycle and retina disease

Fig. 1. Rhodopsin (R) is situated at the intersection of two major pathways, phototransduction/recovery and the retinoid cycle. Rhodopsin, the best studied G protein-coupled receptor, occupies center stage between two physiological pathways: phototransduction/recovery from bleaching (return of activated components to the dark state) and the retinoid cycle (production of 11-cis-retinal) (see Fig. 1). The retinoid cycle has recently gained much attention owing to the fact that multiple genes encoding components with prominent roles in this cycle have been identified, cloned and linked to retina diseases. The intent of the meeting in Ft. Lauderdale (Florida), May 2–3, 2003, was to present a broad overview on the biochemical mechanisms of visual pigment bleaching in light, chromophore recycling, and visual pigment regeneration in the dark. Emphasis was also directed toward retina diseases arising from defects in genes encoding components of the retinoid cycle. These events were thoroughly discussed in eight sessions (see http:// www.visres-interactivemeeting.com/oral.htm) before an audience of over 200 students, researchers and clinicians. Vertebrate phototransduction is initiated by a photochemical reaction whereby 11-cis-retinal bound to its opsin moiety (rhodopsin1⁄4 opsin + 11-cis-retinal) undergoes isomerization to all-trans-retinal producing conformation changes in opsin. The initial events of rhodopsin isomerization, researched intensively for many decades, are the focus of several contributions in this Special Issue (e.g., Janz and Farrens, Pepperberg, Ramon et al.). In vertebrates, restoration of a photosensitive receptor conformation (return to the dark state) requires the formation of 11-cis-retinal from alltrans-retinal via the retinoid cycle. The entire cycle of isomerization and pigment regeneration in humans occurs on a time scale of minutes for rhodopsin, and significantly faster for cone pigments. Reduction of alltrans-retinal to all-trans-retinol takes place in photoreceptor outer segments whereas all other reactions, including isomerization, occur within retinal pigment epithelial cells (RPE) (shown schematically in Fig. 2). The all-trans-retinylidene Schiff base hydrolyzes and all-trans-retinal dissociates from the binding pocket of opsin, yet the molecular steps leading to its release from the opsin-binding pocket remain not fully explained. Removal of all-trans-retinal from the disks