Horizontal Cell Axon Terminals Research Articles

Three-Dimensional Ultrastructure of the Normal Rod Photoreceptor Synapse and Degenerative Changes Induced by Retinal Detachment.

The rod photoreceptor synapse is the first synapse of dim-light vision and one of the most complex in the mammalian CNS. The components of its unique structure, a presynaptic ribbon and a single synaptic invagination enclosing several postsynaptic processes, have been identified, but disagreements about their organization remain. Here, we have used EM tomography to generate high-resolution images of 3-D volumes of the rod synapse from the female domestic cat. We have resolved the synaptic ribbon as a single structure, with a single arciform density, indicating the presence of one long site of transmitter release. The organization of the postsynaptic processes, which has been difficult to resolve with past methods, appears as a tetrad arrangement of two horizontal cell and two rod bipolar cell processes. Retinal detachment severely disrupts this organization. After 7 d, EM tomography reveals withdrawal of rod bipolar dendrites from most spherules; fragmentation of synaptic ribbons, which lose their tight association with the presynaptic membrane; and loss of the highly branched telodendria of the horizontal cell axon terminals. After detachment, the hilus, the opening through which postsynaptic processes enter the invagination, enlarges, exposing the normally sequestered environment within the invagination to the extracellular space of the outer plexiform layer. Our use of EM tomography provides the most accurate description to date of the complex rod synapse and details changes it undergoes during outer segment degeneration. These changes would be expected to disrupt the flow of information in the rod pathway.SIGNIFICANCE STATEMENT Ribbon-type synapses transmit the first electrical signals of vision and hearing. Despite their crucial role in sensory physiology, the three-dimensional ultrastructure of these synapses, especially the complex organization of the rod photoreceptor synapse, is not well understood. We used EM tomography to obtain 3-D imaging at nanoscale resolution to help resolve the organization of rod synapses in normal and detached retinas. This approach has enabled us to show that in the normal retina a single ribbon and arciform density oppose a tetrad of postsynaptic processes. In addition, it enabled us to provide a 3-D perspective of the ultrastructural changes that occur in response to retinal detachment.

Connexin50 couples axon terminals of mouse horizontal cells by homotypic gap junctions.

Horizontal cells in the mouse retina are of the axon-bearing B-type and contribute to the gain control of photoreceptors and to the center-surround organization of bipolar cells by providing feedback and feedforward signals to photoreceptors and bipolar cells, respectively. Horizontal cells form two independent networks, coupled by dendro-dendritic and axo-axonal gap junctions composed of connexin57 (Cx57). In Cx57-deficient mice, occasionally the residual tracer coupling of horizontal cell somata was observed. Also, negative feedback from horizontal cells to photoreceptors, potentially mediated by connexin hemichannels, appeared unaffected. These results point to the expression of a second connexin in mouse horizontal cells. We investigated the expression of Cx50, which was recently identified in axonless A-type horizontal cells of the rabbit retina. In the mouse retina, Cx50-immunoreactive puncta were predominantly localized on large axon terminals of horizontal cells. Electron microscopy did not reveal any Cx50-immunolabeling at the membrane of horizontal cell tips invaginating photoreceptor terminals, ruling out the involvement of Cx50 in negative feedback. Moreover, Cx50 colocalized only rarely with Cx57 on horizontal cell processes, indicating that both connexins form homotypic rather than heterotypic or heteromeric gap junctions. To check whether the expression of Cx50 is changed when Cx57 is lacking, we compared the Cx50 expression in wildtype and Cx57-deficient mice. However, Cx50 expression was unaffected in Cx57-deficient mice. In summary, our results indicate that horizontal cell axon terminals form two independent sets of homotypic gap junctions, a feature which might be important for light adaptation in the retina.

Synaptic Elements for GABAergic Feed-Forward Signaling between HII Horizontal Cells and Blue Cone Bipolar Cells Are Enriched beneath Primate S-Cones

The functional roles and synaptic features of horizontal cells in the mammalian retina are still controversial. Evidence exists for feedback signaling from horizontal cells to cones and feed-forward signaling from horizontal cells to bipolar cells, but the details of the latter remain elusive. Here, immunohistochemistry and confocal microscopy were used to analyze the expression patterns of the SNARE protein syntaxin-4, the GABA receptor subunits α1 and ρ, and the cation-chloride cotransporters NKCC and KCC2 in the outer plexiform layer of primate retina. In macaque retina, as observed previously in other species, syntaxin-4 was expressed on dendrites and axon terminals of horizontal cells at cone pedicles and rod spherules. At cones, syntaxin-4 appeared densely clustered in two bands, at horizontal cell dendritic tips and at the level of desmosome-like junctions. Interestingly, in the lower band where horizontal cells may synapse directly onto bipolar cells, syntaxin-4 was highly enriched beneath short-wavelength sensitive (S) cones and colocalized with calbindin, a marker for HII horizontal cells. The enrichment at S-cones was not observed in either mouse or ground squirrel. Furthermore, high amounts of both GABA receptor and cation-chloride cotransporter subunits were found beneath primate S-cones. Finally, while syntaxin-4 was expressed by both HI and HII horizontal cell types, the intense clustering and colocalization with calbindin at S-cones indicated an enhanced expression in HII cells. Taken together, GABA receptors beneath cone pedicles, chloride transporters, and syntaxin-4 are putative constituents of a synaptic set of proteins which would be required for a GABA-mediated feed-forward pathway via horizontal cells carrying signals directly from cones to bipolar cells.

Ether-à-gogo-related gene (erg1) potassium channels shape the dark response of horizontal cells in the mammalian retina

Postsynaptic to photoreceptors, horizontal cells face prolonged exposure to glutamate in the dark. Therefore, efficient hyperpolarizing mechanisms are crucial to keep horizontal cells within an operating range and to reduce glutamate-induced excitotoxicity. Combining electrophysiology, single-cell reverse transcriptase polymerase chain reaction, and immunocytochemistry, we found that horizontal cell bodies but not their axon terminals express the ether-à-gogo-related gene isoform 1 (erg1) K(+) channel. Erg1-mediated outward currents displayed voltage-dependent activation and C-type inactivation. Recovery from inactivation involved a transient open state. Gating of erg1 channels kept the voltage response to glutamate brief and at physiological amplitudes. With erg1 channels blocked, the response of horizontal cells to the onset of darkness was significantly enhanced. These results indicate a functional dichotomy between horizontal cell bodies and axon terminals in the processing of photoreceptor signals. The dark response thus reflects a finely tuned balance determined by the successive gating of ionotropic glutamate receptors and erg1 channels.

Rod and Cone Contributions to Horizontal Cell Light Responses in the Mouse Retina

Mammalian B-type horizontal cells make contact with both photoreceptor types: the dendrites contact cone photoreceptors, whereas the axon terminal processes contact rods. Despite their distinct synaptic contacts, horizontal cell somata and axon terminals receive a mixture of rod and cone inputs. Interaction of the two photoreceptor systems is essential for adaptation of photoreceptor sensitivity to different levels of background illumination, and horizontal cells play a key role in this adaptation. In this study, we used transgenic mouse lines to examine the contributions of rod and cone photoreceptor inputs to horizontal cell light responses in the mouse retina: rod signals were isolated by recording intracellularly from horizontal cells in a mouse lacking the cone cyclic nucleotide-gated channel, which lacks cone function, and cone signals were assessed using the rhodopsin knock-out mouse, which is a model for pure cone function. We found that both horizontal cell compartments receive a mixture of inputs from both photoreceptor types. To determine whether these inputs arrive via the long axon connecting the compartments or by way of rod-cone gap junctional coupling, we assessed the rod and cone contributions to horizontal cell somatic and axon terminal light responses in the connexin36-deficient mouse retina, which lacks rod-cone coupling. Our results confirm that rods and cones are coupled by connexin36, and suggest that signal transmission along the axon is unidirectional: signals are passed from horizontal cell soma to axon terminal but not from axon terminal to soma.

Dopaminergic modulation of horizontal-cell-axon-terminal receptive field size in the mammalian retina

Receptive fields of gap junction-coupled axon terminals of B-type horizontal cells of isolated rabbit retinae were measured by recording light responses to slit shaped light stimuli at different eccentricities from the recording site. The D1/D5 agonist SKF-38393 and the membrane permeant second messenger 8-bromo-cAMP caused decreases of space constants by 20% while the D1/D5 antagonist SCH-23390 increased space constants by 25%. The results of this study indicate that axon terminal receptive fields of the rabbit retina can be modulated by D1/D5 receptor activation based on a cAMP-mediated mechanism. The data also suggest the presence of endogenous dopamine as an agent for axon terminal receptive field size modulation.

Immunocytochemical localization of three vesicular glutamate transporters in the cat retina.

Vesicular transporters play an essential role in the packaging of glutamate for synaptic release and so are of particular importance in the retina, where glutamate serves as the neurotransmitter for photoreceptors, bipolar cells, and ganglion cells. In the present study, we have examined the distribution of the three known isoforms of vesicular glutamate transporter (VGLUT) in the cat retina. VGLUT1 was localized to all photoreceptor and bipolar cells, whereas VGLUT2 was found in ganglion cells. This basic pattern of complementary distribution for the two transporters among known populations of glutamatergic cells is similar to previous findings in the brain and spinal cord. However, the axon terminals of S-cone photoreceptors were found to express both VGLUT1 and VGLUT2 and some ganglion cells labeled for both VGLUT2 and VGLUT3. Such colocalizations suggest the existence of dual modes of regulation of vesicular glutamate transport in these neurons. Staining for VGLUT2 was also present in a small number of varicose processes, which were seen to ramify throughout the inner plexiform layer. These fibers may represent axon collaterals of ganglion cells. The most prominent site of VGLUT3 immunoreactivity was in a population of amacrine cells; the axon terminals of B-type horizontal cells were also labeled at their contacts with rod spherules. The presence of the VGLUT3 transporter at sites not otherwise implicated in glutamate release may indicate novel modes of glutamate signaling or additional roles for the transporter molecule.

Contribution of connexin26 to electrical feedback inhibition in the turtle retina

The first synaptic integration in the neuronal cascade of vision in vertebrates includes a feedback from horizontal cells to cones by a mechanism yet not fully understood. Recent observations in teleosts suggested an electrical feedback mechanism mediated by connexin26 (Cx26) hemichannels at the terminal dendrites of horizontal cells. By using reverse transcription-polymerase chain reaction and immunoblotting from retinal homogenate, we detected Cx26 mRNA transcripts in the turtle retina and demonstrated that they were translated into protein. Cx26 immunoreactivity was especially prominent in the outer plexiform layer. Subcellularly, immunoreactivity was located mainly between horizontal cell axon terminals and in horizontal cell dendrites forming the lateral elements at the ribbon synaptic complex of the cone pedicle. The label was confined to the horizontal cell membrane flanking the ribbon and was not found on the opposing photoreceptor membrane. No gap junctions at this location are known, so immunosignaling suggested the presence of hemichannels. Their relevance to the feedback mechanism was investigated by intracellular recordings from horizontal cells during application of the hemichannel blocker carbenoxolone. Carbenoxolone hyperpolarized the dark membrane potential by about 25 mV, decreased the amplitudes of responses to full-field light flashes, and suppressed the feedback-induced depolarizing inflexion in the response profile. These physiological findings are compatible with the involvement of hemichannels in the feedback between horizontal cells and cones and support the anatomical findings. Together, these data suggest the presence of an electrical feedback mechanism in the turtle retina, which therefore might be a common mechanism at the first visual synapse in vertebrates.

Axonal neurofilament-H immunolabeling in the rabbit retina.

A monoclonal antibody directed at the multiphosphorylated epitope of axonal neurofilament-H (NF-H) was used to label axon-like fibers in the rabbit retina. NF-H-immunopositive fibers were found in the outer plexiform layer (OPL), inner plexiform layer (IPL), and optic fiber layer (OFL). The morphological characteristics of the labeled processes identified those in the OPL as horizontal cell axons and axon terminals and fibers in the OFL as axons of ganglion cells. The NF-H-positive profiles in the OPL formed a subset of horizontal cell processes labeled for calbindin. In the IPL, NF-H-immunoreactive profiles lay at all levels but were detected most often in the middle strata, 2-4. Occasionally, we observed NF-H-immuoreactive processes emerging from the IPL and entering either the GCL or the inner nuclear layer (INL). The labeled fibers in the IPL were typically very thin, less than 1 microm in diameter, and could often be followed for over 1 mm as they ran laterally across the retina. Cell bodies were never labeled by the immunoserum. To identify the NF-H-immunopositive fibers in the IPL, standard immunocytochemical double-labeling techniques were applied, using antibodies directed against several neurotransmitters or modulators thought to be expressed by axon-bearing amacrine cells. The NF-H-positive processes in the IPL were found to correspond to those labeled for tyrosine hydroxylase, somatostatin, substance P, and NADPH diaphorase activity. However, the NF-H labels did not colocalize with those against the vasoactive intestinal peptide-associated protein PHM27. Our results indicate that putative axons in the retina possess the multiphosphorylated NF-H protein found within classic axons in the central nervous system. These results thus support the idea that certain subtypes of amacrine and horizontal cells maintain true axons in the mammalian retina.

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study.

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.

Endothelial nitric oxide synthase (eNOS) is localized to Müller cells in all vertebrate retinas

The distribution of endothelial nitric oxide synthase immunoreactivity (eNOS-IR) was investigated in the retinas of all phylogenetic vertebrate classes by using a monoclonal eNOS antibody. Confocal light microscopy showed immunoreactive labeling in Müller cells of fish, frog, salamander, turtle, chicken, rat, ground squirrel, and monkey retina. In vascularized retinas (rat, monkey), astrocytes and some blood vessels were also stained. Furthermore, eNOS-IR was localized to axon terminals of turtle and fish horizontal cells. These observations are the first to show the presence of eNOS-IR in Muller glia and horizontal cell structures of the vertebrate retina.

Localization of nNOS in photoreceptor, bipolar and horizontal cells in turtle and rat retinas.

Neuronal nitric oxide synthase (nNOS), an enzyme that synthesizes NO, has been found in the outer retina using light microscopic immunocytochemistry, but its subcellular localization is unknown. We used electron immunocytochemistry to examine nNOS-like immunoreactivity (nNOS-LI) in the outer plexiform layer of turtle and rat retinas. In turtle, nNOS-LI was present in some bipolar and horizontal cell processes at photoreceptor ribbon synapses and at basal junctions between photoreceptors. In rat, nNOS-LI was present in some rod bipolar and B-type horizontal cell axon terminals at rod ribbon synapses. These results indicate that in vertebrates, NO is produced by all of the major nerve cell types in the outer retina at specific synaptic contacts.

3H-adenosine uptake selectively labels rod horizontal cells in goldfish retina

There are four types of horizontal cell in the goldfish retina, three cone- and one rod-type. The neurotransmitter of only one type, the H1 (cone) horizontal cell, has been identified as GABA. 3H-adenosine uptake was examined as a possible marker for the other classes of horizontal cell. Isolated goldfish retinae were incubated in 3H-adenosine (10-40 microCi) in HEPES-buffered saline for 30 min, then fixed, embedded in plastic, and processed for light-microscopic autoradiography (ARG). For double-label immuno/ARG studies, 1-micron-thick sections were processed for GABA postembed immunocytochemistry, then for ARG. 3H-adenosine uptake was localized to cone photoreceptors, presumed precursor cells in the proximal outer nuclear layer, and to a single, continuous row of horizontal cell bodies in the inner nuclear layer. No uptake was localized to the region of horizontal cell axon terminals. 3H-adenosine uptake did not colocalize with GABA-IR in H1 horizontal cells, but it did colocalize with adenosine deaminase immunoreactivity. It is concluded that 3H-adenosine uptake selectively labels rod horizontal cells in the goldfish retina based on position and staining pattern, which are similar to rod horizontal cells stained by Golgi or HRP injection methods. The use of 3H-adenosine uptake may provide a useful tool to study other properties of rod horizontal cells (i.e. development) as well as provide clues as to the transmitter used by these interneurons.

Effects of horizontal cell network architecture on signal spread in the turtle outer retina. experiments and simulations

In the Pseudemys turtle retina five functionally distinct, electrically coupled networks of horizontal cells distribute signals in the outer plexiform layer. These networks differ significantly in their architecture, as determined by intracellular labeling with Neurobiotin after physiological recording and identification. The density of H1 horizontal cells is highest, ranging around 1800 cells/mm 2 at approximately 2.3 mm eccentricity. H1 horizontal cell somata are connected via 6–10 thin, short dendrites. The H1 horizontal cell axon terminal network is composed of thick axon terminals, forming a three-dimensional, sheath-like structure. Networks of coupled H2 and H3 horizontal cells have cell densities of around 210 cells/mm 2 and 350 cells/mm 2 respectively, at the same eccentricity of 2.3 mm. Cell bodies are connected with 6–12 long, thin dendrites. Here we report for the first time H4 horizontal cell networks. Cell density is approximately 970 cells/mm 2 at 2 mm eccentricity, and cell bodies are connected with 6–10 thin, short dendrites. General properties of passive voltage spread were compared for three of these horizontal cell networks using NeuronC. Realistic network architectures were obtained by digitizing the intracellularly labeled networks, respectively. One network obtained from coupled H1 horizontal cell bodies, one from coupled H1 horizontal cell axon terminals, and one from H2 horizontal cells were simulated. These three realistic networks were compared with an artificial, electrically coupled regular triangular network. Passive signal spread in these networks strongly depended on the exact network architecture using otherwise identical parameters. Changes in coupling strength affected signal spread in these networks differently. As in the experimental situation, changes in synaptic conductance influenced signal spread. Some principal effects of extensively coupled horizontal cells on photoreceptor signal processing were simulated with one type of photoreceptor connected by telodendria, synapsing onto an underlying triangular network and receiving feedback synapses. Under certain conditions, spatial information is coded in single photoreceptors. This was also the case in the experimental situation. In the simulation, spatial filter adjustment for optimal spatial coding in photoreceptors can be achieved by changing coupling strength in the horizontal cell network.

Distribution and coverage of A- and B-type horizontal cells stained with Neurobiotin in the rabbit retina.

Both A- and B-type horizontal cells in the rabbit retina were labeled by brief in vitro incubations of the isolated retina in the blue fluorescent dye 4,6-diamino-2-phenylindole. Intracellular injection of Lucifer Yellow into the somata revealed the morphology of the individual cells. Dye-coupling with Lucifer Yellow was seen only between A-type horizontal cells. By contrast, injection of the tracer Neurobiotin showed dye-coupling between both A- and B-type horizontal cells. There also appeared to be coupling between the axon terminals of B-type horizontal cells. The extensive dye-coupling seen following injection of Neurobiotin into a single horizontal cell soma can be used to obtain population counts of each cell type. Staining of large numbers of each cell type across the retina showed that each type increased in number and declined in dendritic diameter as the visual streak was approached, such that relatively constant coverage across the retina was maintained. In the visual streak, A-type horizontal cells numbered 555 cells/mm2 and averaged 120 microns in diameter, compared to 1375 cells/mm2 and 100 microns for B-type horizontal cells. In the periphery, the A- and B-types numbered 250 cells/mm2 and 400 cells/mm2, respectively. The average diameters of the dendritic trees at these locations were 225 microns for the A-type and 175 microns for the B-type. Coverage across the retina averaged almost six for A-type horizontal cells and 8-10 for the B-type. A-type horizontal cells in the visual streak whose elliptical dendritic fields were shown by Bloomfield (1992) to correlate physiologically with orientation bias were shown to be dye-coupled to cells with symmetrical dendritic fields.

The coupling pattern of axon-bearing horizontal cells in the mammalian retina.

In most vertebrate retinae, horizontal cells of the same functional type are homologously coupled through gap junctions. However, ultrastructural, physiological and dye-coupling studies have shown that the axon terminals of horizontal cells in mammalian retinae are not connected by gap junctions. In this study, intracellular injection of the junction-permeant tracer, Neurobiotin, combined with photochromic intensification of the diaminobenzidine (DAB) reaction product, has revealed that the B-type horizontal cells in rabbit retina show strong tracer coupling, both between the dendritic trees and between the axonal arborizations. These findings establish that the coupling pattern of axon-bearing horizontal cells in a mammalian retina in qualitatively similar to the coupling patterns of axon-bearing horizontal cells in non-mammalian retinae.

Membrane currents of horizontal cells isolated from turtle retina

1. Membrane currents of horizontal cells isolated from the retina of the turtle, Pseudemys, were characterized by the whole-cell patch-clamp technique. 2. Four membrane currents were identified: an anomalous rectifier blocked by barium, a transient A-current, a sustained L-type calcium current enhanced by Bay K 8644, and a fast, tetrodotoxin-sensitive sodium current. Each of these four currents was found in both horizontal cell somata and axon terminals. 3. The current-voltage relations of axon terminals and somata were similar, but, in the normal operating range of the cell (-30 to -50 mV), the mean slope resistance of the axon terminal was higher (1.38 G omega) than that of the soma (0.26 G omega). 4. Exposure to either glutamate, kainate, or quisqualate induced a sustained inward current in horizontal cell axon terminals. The reversal potential for this current was -3 mV when tested with voltage steps and +9.1 mV when measured by a voltage ramp. The same horizontal cells were insensitive to N-methyl-D-aspartate. 5. A continuum model was developed to compute the degree of signal transfer between a horizontal cell body and its axon terminal. The model consisted of a network of electrically coupled somata that communicates with a network of electrically coupled axon terminals through the connecting axons. The specific membrane resistances used for the model derived from the patch-clamp measures. 6. We computed the voltage change elicited in either the layer of somata or of axon terminals by a static light stimulus of arbitrary dimensions. The amplitude of a spot response as a function of its radius was given by the weighted sum of two Bessel functions with different space constants. 7. The computed responses of the cell body were dominated by the Bessel function with the smaller space constant, whereas those of the axon terminal depended primarily on the Bessel function with the larger space constant. 8. The model predicts that, in contrast to the findings in teleost retina, there is little signal transfer between the somata and axon terminals of horizontal cell in the turtle retina.

Modulation of connexon densities in gap junctions of horizontal cell perikarya and axon terminals in fish retina: effects of light/dark cycles, interruption of the optic nerve and application of dopamine.

In the fish retina, connexon densities of gap junctions in the outer horizontal cells are modulated in response to different light or dark adaptation times and wavelengths. We have examined whether the connexon density is a suitable parameter of gap junction coupling under in situ conditions. Short-term light adaptation evoked low connexon densities, regardless of whether white or red light was used. Short-term dark adaptation evoked high connexon densities; this was more pronounced in the axon terminal than in perikaryal gap junctions. Under a 12 h red light/12 h dark cycle, a significant difference in connexon densities between the light and the dark period could be established in the gap junctions of the perikarya and axon terminals. Under a white light/dark cycle, only the gap junctions of axon terminals showed a significant difference. Crushing of the optic nerve resulted in an increase in connexon densities; this was more pronounced in axon terminals than in perikarya. Dopamine injected into the right eye of white-light-adapted animals had no effect. However, dopamine prevented the effect of optic-nerve crushing on connexon density. The reaction of axon-terminal gap junctions to different conditions thus resembles that of perikaryal gap junctions, but is more intense. Axon terminals are therefore thought to play an important role in the adaptation process.

Organization and development of horizontal cells in the goldfish retina, I: The use of monoclonal antibody AT101

We have produced and characterized a monoclonal antibody, AT101, which selectively labels both viable and formaldehyde-fixed horizontal cell axon terminals, but not their somas or axons, of the goldfish (Carassius auratus) retina. The antigen recognized by AT101 appears to be a cell surface glycoprotein with a molecular weight of about 35,000 Daltons, and is present exclusively or predominantly in nervous tissues of all vertebrate species examined. We have used AT101 as a probe to analyze immunocytochemically the organization of horizontal cell axon terminals (HCATs) in the adult goldfish retina, and the emergence and maturation of these terminals during retinal development. Because of continued growth at the retinal margin in adult goldfish, there is a peripheral-to-central gradient in the age of cells, with the most mature in the center and the youngest in the periphery. In the center and near periphery of the adult retina, HCATs have a fusiform morphology and form a dense network in the middle and proximal part of the inner nuclear layer. In the far peripheral retina, the axon terminals appear round or ellipsoid. The retina closest to the retinal margin is devoid of AT101 staining, indicating that either HCATs are absent or the antigen recognized by AT101 is not present on HCATs at this stage. A similar sequence of changes in staining pattern is seen during development. Although AT101 staining can first be demonstrated in the larval retina at 1 month after hatching, it appears mostly as punctate structures. At a later stage, there are round or ellipsoid structures that resemble in morphology and location (in the inner nuclear layer) those found in the far peripheral adult retina. Double-labeling experiments with AT101 and antiserum against tubulin also indicate that AT101 labels the HCATs when they first appear during development. These data suggest that the emergence and maturation of HCAT is a late event in retinal development.

Horizontal cell axon terminals in growing goldfish

In the retina of teleost fish, cone horizontal cell axons penetrate the inner nuclear layer, where they enlarge into fusiform terminal swellings. The present study shows that horizontal cell axon terminals enlarge disproportionately during postembryonic growth of the retina in juvenile and adult goldfish: the relative volume of axon terminals increases almost 20-fold, while the volume of the entire retina increases only about fourfold during a 2–3-yr period. The enlarging axon terminals fill in the gaps created as the numerical density of nuclei in the inner nuclear layer falls. Horizontal cell axon terminals are thought to participate in cone-dominated visual pathways, although their precise role is unclear. The results of this study suggest that a comparison of horizontal cell function in small and large fish might help to resolve this issue.