White perch horizontal cells in culture: Methods, morphology and process growth

Spatial Distribution of Potential in a Flat Cell: Application to the Catfish Horizontal Cell Layers

1. Horizontal cells of the cat retina were isolated by enzymatic dissociation. Two types of horizontal cells were identified: the axonless (A-type) horizontal cell having four to six thick, long (approximately 100 microns) dendrites, and the short-axon (B-type) horizontal cell having many (> 5) fine, short (approximately 30 microns) dendrites. 2. Membrane properties of isolated horizontal cells were analyzed under current-clamp and voltage-clamp conditions. In the A-type cell, the average resting potential was -55 mV and the mean membrane capacitance was 110 pF, whereas values in the B-type cell were -58 mV and 40 pF, respectively. The A-type cell showed long-lasting Ca spikes, but B-type cells had no Ca spikes. 3. Five types of voltage-dependent ionic currents were recorded: a sodium current (INa), a calcium current (ICa), and three types of potassium currents. Potassium currents consisted of potassium current through the inward rectifier (Ianomal), transient outward potassium current (IA), and potassium current through the delayed rectifier (IK(v)). INa was recorded only from A-type cells. Other currents were recorded from both types of cells. 4. INa activated when cells were depolarized from a holding potential (Vh) of -95 mV, and it was maximal at -25 mV. This current was blocked by tetrodotoxin. Approximately half of the A-type cells had INa, but no B-type cell had this current. 5. L-type ICa, an inward-going sustained current, was activated with depolarization more positive than -25 mV. Current amplitude reached a maximal value near 15 mV and became smaller with further depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

There are two types of horizontal cells, the luminosity and the chromaticity cells, in the retina of the kissing gourami, Helostoma rudolfi. Luminosity cells occupy the outermost layer proximal to the receptor terminals, whereas chromaticity cells form a layer proximal to the layer of luminosity cells. Neither type of cell has axons. Responses were evoked by light from red and green light-emitting diodes. The two stimuli were modulated either by a pulsatile or a white-noise signal. The luminosity cell always produced a hyperpolarizing response. The chromaticity cell produced a hyperpolarizing response when stimulated by only one color. However, in the presence of a steady or modulated green input, a red stimulus produced a depolarizing response. Such chromaticity cells were similar to the (spectral) biphasic chromaticity horizontal cells observed in other retinae. The depolarizing phase of the red response was produced by the balance of intensity of the two inputs, red and green. We used white-noise methodology to identify the dynamics of the horizontal cell's modulation response by taking advantage of the fact that a Wiener kernel is a measure of a cell's incremental sensitivity, which includes its response dynamics. Under all conditions, a steady state modulation response by both luminosity and chromaticity cells always was related linearly to the input modulation. The average mean square error (MSE) of the model predicted by the first-order kernel was approximately 8% for both luminosity (n = 116) and chromaticity (n = 23) cells. In some cases, the MSE was a few percent even when the peak-to-peak response amplitude was nearly 30 mV. The ratio of inputs from red and green cones to both types of horizontal cells was variable; the major input for luminosity cells came from red cones, whereas the major input for chromaticity cells came from green cones. First-order kernels generated by the major input were robust in terms of waveform in the sense that the waveform remained unchanged whether or not there was a steady or modulated illumination by the opposing color. The results reported here do not address the question of the neural circuitry that generates horizontal cell responses, in particular, the depolarizing response. However, whatever that circuitry might be, the high degree of linearity of the modulation response by both types of cell under various stimulus conditions imposes restrictions on the performance of any proposed model as well as on mechanisms that underlie the generation of the horizontal cell response.

Anatomy and physiology of horizontal cells in layer 5b of the chicken optic tectum

Horizontal cells from the retinas of white perch were isolated and maintained in cell culture for 3 days to 3 wk. Four morphologically distinct types of horizontal cells could be identified in culture and were labeled types H1, H2, H3, and H4. Whole-cell patch-clamp techniques were used to study the ionic currents present in the four cell types. In all cells, depolarizing commands above threshold elicited a fast-inward current followed by an outward current. The fast-inward current was abolished by tetrodotoxin (TTX) or 0 Na+ Ringer's, indicating the current was carried by Na+. In H1, H2, and H3 cells, the outward current, carried by K+, consisted of two components: a transient current (IA), blockable with 4-aminopyridine (4-AP), tetraethylammonium (TEA), or intracellular cesium and a sustained current that could be blocked with TEA. The H4 cell had only the sustained current. An inward rectifying K+ current (anomalous rectifier) was observed in the four cell types. The current was sensitive to the extracellular K+ concentration. Its activation showed two components: an instantaneous component and a slower component. The slow component becomes faster with greater hyperpolarizations. The four cell types possessed a small, sustained Ca2+ current that, under normal conditions, was masked by the inward Na+ current and outward K+ currents.

Retinas of ordinary and black moor varieties of goldfish (Carassius auratus) were prepared by the Golgi method, mounted flat or sectioned vertically, and studied in the light microscope. Three types of horizontal cells whose dendrites contact only cones, and one type whose dendrites contact only rods, were observed. The cone horizontal cells (Cajal's "external horizontal cells") all have slender axons which descend gradually to the inner nuclear layer and terminate there in long, fusiform expansions (Cajal's "internal horizontal cells"). The thin and thick portions of the axons, as well as the perikarya of the horizontal cells, bear small numbers of straight, horizontally-directed, knobby filamentous appendages which may be sites of synaptic contact. The cone horizontal cell axons in goldfish, unlike those in higher vertebrates, do not terminate in contact with synaptic endings of photoreceptor cells, but in proximity to cells and processes deep in the inner nuclear layer. Axons have not yet been demonstrated on rod horizontal cells in goldfish.

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

Variety of horizontal cell gap junctions in the rabbit retina

Quantification of Horizontal Cells in the Domestic Cat Retina

The vertebrate retina consists of five types of neurons orderly arranged in three layers. Signals received by the photoreceptor mosaic are relayed to bipolar cells, and then forwarded to retinal ganglion cells. The axon of retinal ganglion cells send the visual signal processed in the retina to the higher CNS. The neuronal chain made of photoreceptors, bipolar cells and ganglion cells provides the most direct signal pathway in the retina. Two types of interneurons, horizontal cells and amacrine cells, are believed to make an inhibitory pathway connecting the above-mentioned direct routes laterally. In this way, horizontal and amacrine cells contribute to the formation of receptive field surrounds which antagonize with the receptive field center.

The effects of prolonged (greater than 2 hr) darkness and background illumination on the light responsiveness of cone horizontal cells were examined in isolated, superfused white perch retinas. In retinas from fish maintained in complete darkness for more than 2 hr, cone horizontal cells had a resting membrane potential of about -18 mV, and they generated only slow, low-amplitude (3-4 mV) responses even when stimulated with bright flashes. Following the presentation of dim background light, the cone horizontal cells slowly hyperpolarized and thereafter remained at a more hyperpolarized level (about -25 mV). Concurrently, their light responses were dramatically enhanced in size, and response amplitudes to bright flashes eventually increased to about 50 mV. This was accompanied by noticeable changes in response waveforms; following light exposure, the responses became faster and showed initial on-transients. The increase in cone horizontal cell responsiveness was graded with intensity of the background light. A similar enhancement in response amplitudes of cone horizontal cells occurred after presenting bright flashes repetitively at intervals of 9 sec. This background sensitization phenomenon was observed in both L- and C-type cone horizontal cells. When light-sensitized isolated retinas were maintained in darkness for long periods (greater than 30 min), the light responsiveness of cone horizontal cells gradually decreased. The changes in membrane potential and response waveform were opposite to those that occurred when prolonged dark-adapted retinas were exposed to background lights; the cells depolarized by 5-7 mV and light-evoked responses became slower. Effects of background illumination on rod-driven horizontal cells were examined as well. Rod horizontal cells were about 2 log units more sensitive to white light than were cone horizontal cells. When recorded in prolonged dark-adapted retinas, rod horizontal cells showed large responses (approximately 50 mV), which could not be further enhanced by background illumination. Cone horizontal cell responsiveness in the carp retina was also depressed by prolonged darkness and increased by illumination in a fashion similar to that observed in the white perch retina. The change in response amplitude was only about 2-fold in carp compared with 5- to 10-fold changes observed in white perch. These results indicate that the light responsiveness of cone horizontal cells in the teleost retina is suppressed in prolonged darkness and that background lights release the cells from suppression.(ABSTRACT TRUNCATED AT 400 WORDS)

We examined the identities of horizontal cell (HC) lateral components in cone terminals and the expression of glutamate receptors on the tips of HC dendrites. We injected A-type horizontal cells (AHCs) with neurobiotin and demonstrated that neurobiotin labeled completely all AHCs within a patch of retina. We converted neurobiotin by using diaminobenzidine and considered labeled processes to be from AHCs and unlabeled processes to be from B-type horizontal cells (BHCs). Three possible combinations of HC dendrites could exist in cone pedicles: both lateral components originating from AHCs, both from BHCs, or one from an AHC and the other from a BHC. EM observations revealed that a majority of cone terminals contained about equal numbers of lateral components originating from each of the two types of HCs and that each of the three possible combinations was present in equal numbers. Localization of different types of glutamate receptors on HC dendritic tips showed that 55% of AHC dendritic tips expressed AMPA receptors and 30% expressed kainate receptors, whereas, in the case of BHCs, 22% of dendritic tips expressed AMPA receptors and 33% expressed kainate receptors. This study suggests that cone photoreceptors feed the light signal equally into networks of AHCs and BHCs and that differential expression of AMPA/kainate receptors by different HCs could account for different functions.

Biphasic C type horizontal cells of the retina of crucian carp were intracellularly stained with horseradish peroxidase (HRP) and quantitatively studied with computer-aided three-dimensional reconstruction techniques. Several parameters characterizing their morphology were determined and compared among different subtypes of horizontal cells. The results show that both cell body area and horizontal extension are proper parameters by which different subtypes of biphasic horizontal cells can be clearly classified.

L-glutamate, the photoreceptor neurotransmitter, depolarizes horizontal cells and OFF bipolar cells by ionotropic AMPA-glutamate receptors. The AMPA-receptor subunit (GluR4) is localized to dendrites of OFF bipolar cells in goldfish retina. Here, we used immunohistochemical techniques to identify AMPA-receptor subunits on horizontal cell dendrites. A monoclonal antibody against rat GluR2, with high sequence homology to the recently cloned goldfish GluR2a receptor, was used for light- and electron-microscopical immunocytochemistry. Light- and dark-adapted retinas were analyzed, with no major difference in results. GluR2-immunoreactivity (IR) was restricted to a narrow band in the outer plexiform layer, in which it appeared as bright dome-shaped structures amidst numerous puncta. At the ultrastructural level, GluR2-IR was found in horizontal cell dendrites that invaginated cones and rods. Dendrites of OFF bipolar cells were not labeled. GluR2-IR was present mostly in horizontal cell dendrites that were the lateral elements of the triad, rather than in dendrites that were the central elements. In light-adapted retinas, GluR2-IR was found in many horizontal cell spinules. GluR2-IR was observed, on occasion, in a mixed rod/cone (Mb) ON bipolar cell process that innervated rod spherules. Verification of the Mb ON bipolar cell was made by protein kinase C and metabotropic mGluR1alpha immunolabeling. The presence of GluR2-IR in lateral elements suggests that lateral horizontal cell dendrites are postsynaptic to cones rather than only sites of feedback inhibition. All horizontal cell types express the GluR2 subunit, uniquely differentiating themselves from OFF bipolar cells that express the GluR4 subunit. This differentiation most likely has a major influence on the glutamate pharmacology and response kinetics of these cell types to glutamate.

Intracellular potassium activities of horizontal cells and extracellular potassium activity in isolated retinae of a cyprinid fish