The vertebrate retina receives, processes and transforms visual information. Light absorbed in photoreceptors is transformed into electrical signa.ls that evoke light modulated release of glutamate onto horizontal and bipolar cells (HCs and BCs). HCs provide lateral feedback regulating transmitter release from photoreceptors according to conditions of ambient illumination, while BCs transfer light signals forward to amacrine cells (ACs) and ganglion cells (GCs). ACs are retinal interneurons with mainly inhibitory and modulatory roles. GCs transform light signals into trains of action potentials that propagate through the optic nerve to brain visual centers. As images pass through retinal circuitry, they are decomposed into component parts, so that at the retinal output specialized sets of GCs extract different components of the image. Some signal highlights, others shadows, movement, direction or color. Signal processing within the retina is achieved by sets of neural circuits. These are composed of patterns of connections among neurons, and specialized neurotransmitter to neurotransmitter receptor interactions. The research program studies the relationships between receptor expression on retinal neurons, the neural circuitry of the retina, and retinal information processing tasks. Zebrafish provides a model of human genetic disease. Through studies of this model, genetic and other perturbations of human vision may be better understood. We examine zebrafish retinal function using acutely dissociated retinal neurons, light responses from in vitro eyecup preparations, and zebrafish retinal slices. Using the voltage probe (oxonol, DiBaC4(5)) to study glutamate responses in dissociated zebrafish retinal HCs we have found both short and long term effects on membrane potential. The short-term effect is classical excitation. The long-term effect is a post excitatory restoration of membrane potential (after-hyperpolarization or AHP). AHP also occurs in OFF-type BCs but not in ON-type BCs. It peaks about 5 min after glutamate application. Glutamate antagonists (CNQX) block AHP. Some glutamate agonists (kainate, AMPA, (S)-5-Fluorowillardiine) evoke the response whereas others (NMDA, D-aspartate, SYM 2081, DL-AP4) do not. This pattern suggests AMPA receptor activation. Some HCs are tonically depolarized. Glutamate evokes only AHP signals in these cells. Resting potentials are restored by nifedipine in tonically depolarized cells, suggesting a sustained, depolarizing action of L-type Ca2+ channels. AHP is not blocked by the L-type Ca2+ channel-blocker nifedipine, however, and it is insensitive to [Cl-]o. AHP is blocked by [Li+]o substitution for [Na+]o and by ouabain. This suggests that Na+ entering through ionotropic AMPA channels stimulates Na+, K+ -ATPase. The electrogenic action of the ATPase restores membrane potential, generating the AHP response. Patterns of ATPase immunoreactivity support localization in the outer plexiform layer (OPL) as cone pedicles, HCs, and BCs are strongly labeled. Persistent stimulation of distal retina by photoreceptor glutamate may induce increased expression and activity of Na+, K+ -ATPase, with a consequent impact on distal glutamate responses. Heightened ATPase activity may be one of several adaptive mechanisms protecting distal retinal neurons against excitotoxicity from tonically released photoreceptor glutamate. Cell structure, a key element in discerning retinal circuits, is beautifully delineated in the zebrafish model. Neurons in retinal slices can be stained in isolation shotgun fashion by spraying DiI coated microcarriers onto the cut surface with a gene gun. This is the Diolisitic technique. Diffusion of the lipophilic tracer along membranes reveals the shapes of individual neurons. In a study of 300 stained zebrafish retinal neurons different subtypes of HCs, BCs and ACs were observed. Based on cell body shape, and the presence or absence of an axon, 3 HC morphologies were identified corresponding to stellate and elongate types earlier seen in cell culture. Based on branching patterns of axons and dendrites within the retinal inner plexiform layer 17 BC morphologies and 7 AC morphologies were discerned. Cells with ON-type (branching in IPL sublamina b), OFF-type (branching in sublamina a) morphological signatures were about equally prevalent. Mixed a/b branching patterns were also observed. In previous studies using Lucifer-dye filled patch electrodes 13 morphological types of bipolar cell were identified. There is substantial agreement on morphological types between the two studies. Whole-cell patch recording and puff pipette techniques have identified glutamate receptor mechanisms on the BC dendrites of many of these types showing a distribution of 3 basic glutamate receptor types: AMPA/kainate (OFF cells), mGluR6 (ON cells) and glutamate-gated chloride currents (Iglu, ON cells). Diolisitic studies, however suggest that in addition to 3 distinct strata within sublamina a of the IPL, and 2 within sublamina b, there is also a third stratum of sublamina b, intermixed with the retinal ganglion cell layer. ARPE-19 cells are a self-immortalized human retinal pigment ethelial cell line that transforms into neural like morphologies with processes and growth cones after treatment with the vitamin A derivative phenretinide. Studies are currently underway to examine expression of neurotransmitter receptors in these cells.
| Connaughton, V P; Graham, D; Nelson, R (2004) Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina. J Comp Neurol 477:371-85 |
| Kolb, H; Nelson, R; Ahnelt, P et al. (2001) Cellular organization of the vertebrate retina. Prog Brain Res 131:3-26 |
| Nelson, R; Janis, A T; Behar, T N et al. (2001) Physiological responses associated with kainate receptor immunoreactivity in dissociated zebrafish retinal neurons: a voltage probe study. Prog Brain Res 131:255-65 |
| Connaughton, V P; Nelson, R (2000) Axonal stratification patterns and glutamate-gated conductance mechanisms in zebrafish retinal bipolar cells. J Physiol 524 Pt 1:135-46 |
| Nelson, R; Schaffner, A E; Li, Y X et al. (1999) Distribution of GABA(C)-like responses among acutely dissociated rat retinal neurons. Vis Neurosci 16:179-90 |
