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Horizontal cells invest retinal capillaries in the tree shrew Tupaia belangeri.

Retinal capillaries were studied by transmission electron microscopy, immunohistochemistry and lectin histochemistry in the adult tree shrew Tupaia betangeri. In capillaries from all four vascular layers, adjacent endothelial cells were connected by tight junctions. Up to three layers of pericyte processes were embedded in the subendothelial basal lamina. However, pericytes frequently contacted the endothelial cells. In the innermost vascular layer (capillary layer 4), S-100-immunopositive astrocytes and the vitreal processes of S-100-immunopositive Muller cells entirely ensheathed the capillary basal lamina. However, capillaries revealing an incomplete macroglial investment were observed in the outer vascular layers, predominantly in capillary layers 1 and 2. In sections of capillaries located between the inner nuclear layer and the outer plexiform layer (capillary layer 1), these "gaps" were filled with the perikarya and electron-lucent processes of horizontal cells that ensheathed up to approximately nine tenths of the capillary circumference. Horizontal cells were identified by ultrastructural criteria. They were distinct from microglial cells by not being reactive for Griffonia simplicifolia isolectin-B4. In Tutpaia, vessel-contacting horizontal cells reside in a position reported to be occupied by the processes of Müller cells in other mammals. Current concepts of retinal function and pathology, which are based on the assumption that retinal vessels are strictly isolated from retinal neurons, at least in Tupaia, might deserve reconsideration.

Animals↗

Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina.

The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of gamma-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca(2+)-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells.

Adaptor Proteins, Vesicular Transport↗

Receptive field properties of rod-driven horizontal cells in the skate retina.

The large receptive fields of retinal horizontal cells result primarily from extensive intercellular coupling via gap (electrical) junctions; thus, the extent of the receptive field provides an index of the degree to which the cells are electrically coupled. For rod-driven horizontal cells in the dark-adapted skate retina, a space constant of 1.18 +/- 0.15 mm (SD) was obtained from measurements with a moving slit stimulus, and a comparable value (1.43 +/- 0.55 mm) was obtained with variation in spot diameter. These values, and the extensive spread of a fluorescent dye (Lucifer Yellow) from the site of injection to neighboring cells, indicate that the horizontal cells of the all-rod retina of skate are well coupled electrically. Neither the receptive field properties nor the gap-junctional features of skate horizontal cells were influenced by the adaptive state of the retina: (a) the receptive field organization was unaffected by light adaptation, (b) similar dye coupling was seen in both dark- and light-adapted retinae, and (c) no significant differences were found in the gap-junctional particle densities measured in dark- and light-adapted retinas, i.e., 3,184 +/- 286/microns 2 (n = 8) and 3,073 +/- 494/microns 2 (n = 11), respectively. Moreover, the receptive fields of skate horizontal cells were not altered by either dopamine, glycine, GABA, or the GABAA receptor antagonists bicuculline and picrotoxin. We conclude that the rod-driven horizontal cells of the skate retina are tightly coupled to one another, and that the coupling is not affected by photic and pharmacological conditions that are known to modulate intercellular coupling between cone-driven horizontal cells in other species.

Animals↗

Developmental improvement in the regularity and packing of mouse horizontal cells: implications for mechanisms underlying mosaic pattern formation.

The present investigation has sought to determine whether the population of retinal horizontal cells undergoes an increase in the precision of its mosaic patterning during postnatal development, and if so, whether this increase is compatible with three different mechanistic accounts of retinal mosaic formation. Horizontal cells were labeled with antibodies to neurofilaments or calbindin at different developmental stages, and then visualized in retinal wholemounts. Multiple fields were sampled from each retina to determine horizontal cell density, while the X-Y coordinates of each cell in a field were determined. An estimate of total horizontal cell number was calculated for each retina, while the Voronoi domain regularity index and the packing factor were computed for each field. Two strains of mice showing a two-fold difference in the size of their horizontal cell population in maturity were sampled, C57BL/6J and A/J. Horizontal cell number in C57BL/6J was approximately twice that observed in A/J at all postnatal stages, with neither strain showing an effect of age on horizontal cell number. In both strains, however, the Voronoi domain regularity index and the packing factor were significantly lower at P-1 relative to later developmental stages. These results show that accounts of mosaic formation proposing the selective death of irregularly positioned cells, or the periodic occurrence of fate-determining events, are insufficient to establish the final patterning achieved by horizontal cells. Rather, they support the hypothesis that tangential dispersion enhances mosaic patterning during postnatal development.

Aging↗

Neurite outgrowth from bipolar and horizontal cells after experimental retinal detachment.

PURPOSE: To study the responses of horizontal cells and rod bipolar cells, the second-order neurons in the retina, to the degeneration induced by experimental retinal detachment. METHODS: Retinas from the eyes of domestic cats were examined 1, 3, 7, and 28 days after detachment using immunocytochemical and electron microscopic analyses. Retinal sections were labeled with antibodies to synaptophysin, calbindin D, and protein kinase C (PKC), proteins that serve as markers for synaptic terminals, horizontal cells, and rod bipolar cells, respectively. RESULTS: Beginning 1 day after detachment, the outer plexiform layer becomes disorganized and synaptophysin-labeled photoreceptor terminals are detected among the cell bodies of photoreceptors in the outer nuclear layer (ONL). At the same time, horizontal and rod bipolar cell processes grow into the ONL. In some cases, these processes contact photoreceptor terminals that have withdrawn deep into the ONL. Double-labeling experiments with antibodies to glial fibrillary acidic protein (Müller cell labeling) and phosphodiesterase gamma (cone labeling) demonstrate that the calbindin D- and PKC-positive neurite outgrowths are not derived from either Müller cells or cone photoreceptors. CONCLUSIONS: Horizontal and rod bipolar cell processes lengthen after retinal detachment, perhaps in response to a withdrawal of their presynaptic targets, the photoreceptor synaptic terminals. This apparent attempt to maintain synaptic contact after injury demonstrates a plasticity in the adult retina that may be of importance for the recovery of vision in human patients.

Animals↗

A circadian clock regulates rod and cone input to fish retinal cone horizontal cells.

In the vertebrate retina, the light responses of post-receptor neurons depend on the ambient or background illumination. Using intracellular recording, we have found that a circadian clock regulates the light responses of dark-adapted fish cone horizontal cells. Goldfish were maintained on a 12-hr light/12-hr dark cycle. At different times of the day or night, retinas were superfused in darkness for 90 min ("prolonged darkness"), following which horizontal cells were impaled without the aid of any light flashes. In some of the experiments, fish were kept in constant darkness for 3-48 hr prior to surgery. After prolonged darkness during the night, but not during the day, the light responses of L-type cone horizontal cells resembled those of rod horizontal cells with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Following light sensitization during the night and day, the light responses of rod and cone horizontal cells were clearly different with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Under conditions of constant darkness for two full light/dark cycles, average responses of cone horizontal cells to a bright light stimulus during the subjective day were greater than during the subjective night. Prior reversal of the light/dark cycle reversed the 24-hr rhythm of cone horizontal cell responses to bright lights. In addition, following one full cycle of constant darkness, average cone horizontal cell spectral sensitivity during the subjective night closely matched that of rod horizontal cells, whereas average cone horizontal cell spectral sensitivity during the subjective day was similar to that of red (625 nm) cones. These results indicate that the effects of dark adaptation depend on the time of day and are regulated by a circadian clock so that cone input to cone horizontal cells predominates in the day and rod input predominates in the night.

Animals↗

Protein content and cAMP-dependent phosphorylation of fractionated white perch retina.

In the retinas of teleost fish dopamine, released from interplexiform cells, modulates synaptic transmission at both the chemical and electrical synapses of retinal horizontal cells. This modulation is due to activation of adenylate cyclase and phosphorylation by protein kinase A, perhaps of the synaptic ion channel proteins themselves. In this study we have fractionated the white perch retina by Percoll density gradient centrifugation in order to identify proteins which coenrich with horizontal cells. In addition we have tested retinal fractions for phosphorylation by native cAMP-dependent kinase. Our findings indicate that there are at least 3 proteins of molecular weights 28, 43/44 and 50 kDa which coenrich with horizontal cells and 3 proteins of 30/31 kDa, 35 kDa (putative rhodopsin) and 48 kDa (putative arrestin) which coenrich with photoreceptor fractions. The 43/44 kDa phosphoprotein is a target for cAMP-dependent protein phosphorylation and thus is apparently an element of the dopaminergic modulatory pathway in perch horizontal cells.

Animals↗

Quisqualate and L-glutamate inhibit retinal horizontal-cell responses to kainate.

Currents elicited by L-glutamate and the related agonists quisqualate and kainate were analyzed under voltage clamp in isolated goldfish horizontal cells, using the whole-cell recording configuration of the patch-clamp method [Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100]. These currents resulted from an increase in cationic conductance and were indistinguishable from one another in terms of reversal potential (approximately equal to 0 mV) and apparent elementary conductance (2-3 pS). The power-density spectra of the noise increases produced by each agonist were fit by the sum of two Lorentzian curves having similar cutoff frequencies (tau 1 approximately equal to 5 msec, tau 2 approximately equal to 1 msec), but the relative power of these components were different for quisqualate and glutamate than for kainate. Moreover, the responses to high doses of either quisqualate or glutamate rapidly faded, whereas the responses to kainate did not. Finally, quisqualate and glutamate produced an inhibition of responses to kainate which appeared to be uncompetitive. Kainate, quisqualate, and in our preparation, glutamate appear to activate channels different than those activated by N-methyl-D-aspartate in other preparations. At least some of the effects of quisqualate and glutamate appear to be mediated by receptors bound by kainate.

Animals↗

Evidence for hemi-gap junctional channels in isolated horizontal cells of the skate retina.

Prolonged depolarization of isolated, voltage-clamped skate retinal horizontal cells produces an outward current that exhibit a late onset and develops slowly with time. This current, which we refer to as the Q-current, is associated with an increase in membrane conductance, and is present when other voltage-gated conductances have been pharmacologically blocked. The reversal potential for the Q-current, obtained using tail current analysis, was close to 0 mV. The magnitude of the current was greatly reduced by superfusion with 25 mM acetate, and by 4 mM cobalt chloride, 2 mM 1-octanol, and a saturated solution of the general anesthetic halothane. In addition, the low-molecular weight fluorescent dye Lucifer yellow, applied extracellularly, entered the cells during activation of the Q-current, whereas a 3 kD dextran-fluorescein complex did not cross the cell membrane. The effects of divalent cations, the non-specific nature of the ionic current suggested by its reversal potential, the entry of Lucifer yellow, and the ability of acetate, halothane, cobalt, and octanol to block the current lead us to hypothesize that the Q-current results from the opening of hemi-gap junctional channels that mediate electrical coupling between skate horizontal cells.

Animals↗

Morphological types of horizontal cell in rodent retinae: a comparison of rat, mouse, gerbil, and guinea pig.

Retinal horizontal cells of four rodent species, rat, mouse, gerbil, and guinea pig were examined to determine whether they conform to the basic pattern of two horizontal cell types found in other mammalian orders. Intracellular injections of Lucifer-Yellow were made to reveal the morphologies of individual cells. Immunocytochemistry with antisera against the calcium-binding proteins calbindin D-28k and parvalbumin was used to assess population densities and mosaics. Lucifer-Yellow injections showed axonless A-type and axon-bearing B-type horizontal cells in guinea pig, but revealed only B-type cells in rat and gerbil retinae. Calbindin immunocytochemistry labeled the A- and B-type populations in guinea pig, but only a homogeneous regular mosaic of cells with B-type features in rat, mouse, and gerbil. All calbindin-immunoreactive horizontal cells in the latter species were also parvalbumin-immunoreactive; comparison with Nissl-stained retinae showed that both antisera label all of the horizontal cells. Taken together, the data from cell injections and the population studies provide strong evidence that rat, mouse, and gerbil retinae have only one type of horizontal cell, the axon-bearing B-type, whereas the guinea pig has both A- and B-type cells. Thus, at least three members of the family Muridae differ from other rodents and deviate from the proposed mammalian scheme of horizontal cell types. The absence of A-type cells is apparently not linked to any peculiarities in the photoreceptor populations, and there is no consistent match between the topographic distributions of the horizontal cells and those of the cone photoreceptors or ganglion cells across the four rodent species. However, the cone to horizontal cell ratio is rather similar in the species with and without A-type cells.

Animals↗

Unexpected presence of neurofilaments in axon-bearing horizontal cells of the mammalian retina.

In several mammals only one of the two types of retinal horizontal cell, the axonless A-type, appears to express neurofilaments. Neurofilament immunostaining of rodent retinas reveals a horizontal cell plexus that has previously been interpreted as belonging to A-type cells. Our intracellular Lucifer yellow injections strongly suggest that there are no A-type horizontal cells in rat and gerbil. Counterstaining of dye-injected cellular structures with a neurofilament antibody directly shows that the axon terminal systems of the axon-bearing B-type horizontal cells contain neurofilaments. These unexpected findings explain and reinterpret the neurofilament plexus in rodent retinas. In contrast, Lucifer yellow injections in guinea pig retina reveal both A- and B-type horizontal cells, showing that horizontal cell types are not uniform among rodents. In the guinea pig retina both A-type cells and B-type axon terminal systems contain neurofilaments.

Animals↗

Dopamine enhances a glutamate-gated ionic current in OFF bipolar cells of the tiger salamander retina.

The transmitter glutamate is thought to be used by all vertebrate photoreceptors to drive the second-order neurons of the retina, horizontal and bipolar neurons. Dopamine, an endogenous retinal neurotransmitter localized to amacrine and interplexiform cells, has previously been shown to enhance glutamate-gated currents in retinal horizontal cells. In the present study we demonstrate that bipolar cells, like horizontal cells, possess glutamate receptors that are modulated by dopamine. We then identify some components of the pathway through which dopamine acts. We used whole-cell patch recording to measure how bath-applied dopamine modulated the currents elicited by puffs of transmitter solutions at bipolar cell dendrites. Excitatory amino acid-gated currents were evoked by pressure ejecting 1 mM glutamate or 10 microM kainate for 40 msec through a micropipette positioned at the dendrites of bipolar cells. Bath-applied dopamine (20 microM) enhanced the response to glutamate in OFF bipolar cells in the retinal slice by 40% and in isolated OFF bipolar cells by 65%. We also explored the components of the intracellular pathway mediating this modulation. Response enhancement was blocked by the D1 receptor antagonist SCH23390, but not by the D2 receptor antagonist spiperone, suggesting that the enhancement by dopamine is mediated by a D1 receptor. GDP-beta-S, a G-protein inactivator, blocked the enhancing action of dopamine, suggesting that the D1 receptor activated a G-protein to enhance the glutamate-gated current. Both 8-(4-chlorophenylthio)adenosine, a cAMP analog, and the addition of the catalytic subunit of protein kinase A (PKA) to the recording pipette enhanced glutamate-gated currents, while H-7, a PK inactivator, and PKI20amide, a PKA-specific inhibitor, blocked the enhancing action of dopamine. These data suggest that dopamine acts at D1 receptors in the dendrites of bipolar cells to activate adenyl cyclase, which through cAMP enhances a glutamate-gated current in bipolar cell dendrites. Thus, dopamine may modulate synaptic transmission from photoreceptors to OFF bipolar cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Rod bipolar cells and horizontal cells form displaced synaptic contacts with rods in the outer nuclear layer of the nob2 retina.

The nob2 mouse carries a null mutation in the Cacna1f gene, which encodes the pore-forming subunit of the L-type calcium channel, Ca(v)1.4. The loss of the electroretinogram b-wave in these mice suggests a severe reduction in transmission between photoreceptors and second-order neurons in the retina and supports a central role for the Ca(v)1.4 calcium channel at photoreceptor ribbon synapses, to which it has been localized. Here we show that the loss of Ca(v)1.4 leads to the aberrant outgrowth of rod bipolar cell dendrites and horizontal cell processes into the outer nuclear layer (ONL) of the nob2 retina and to the formation of ectopic synaptic contacts with rod photoreceptors in the ONL. Ectopic contacts are predominantly between rods and rod bipolar cells, with horizontal cell processes also present at some sites. Ectopic contacts contain apposed pre- and postsynaptic specializations, albeit with malformed synaptic ribbons. Cone photoreceptor terminals do not participate in ectopic contacts in the ONL. During retinal development, ectopic contacts appear in the days after eye opening, appearing progressively farther into the ONL at later postnatal stages. Ectopic contacts develop at the tips of rod bipolar cell dendrites and are less frequently associated with the tips of horizontal cell processes, consistent with the adult phenotype. The relative occurrence of pre- and postsynaptic markers in the ONL during development suggests a mechanism for the formation of ectopic synaptic contacts that is driven by the retraction of rod photoreceptor terminals and neurite outgrowth by rod bipolar cell dendrites.

Animals↗

Identification and localization of connexin26 within the photoreceptor-horizontal cell synaptic complex.

Connexin26 (Cx26) is a member of the family of integral membrane proteins that normally form intercellular gap junctional channels. We have used Western blotting, immunofluorescence, immunoelectron microscopy, and single-cell reverse-transcriptase polymerase chain reaction amplification (RT-PCR) to analyze the expression and cellular localization of Cx26 in the carp retina. In the outer plexiform layer, strong clustered Cx26 immunolabeling was concentrated at and restricted to the terminal dendrites of horizontal cells. Single-cell RT-PCR confirmed the expression of Cx26 in carp retinal horizontal cells. 248-bp fragments amplified from cDNAs of four different horizontal cells were cloned and each nucleotide sequence encodes a protein fragment (AA 104-185) with highly significant homology to rat and mouse Cx26. Immunoelectron microscopy revealed that only the invaginating dendrites of horizontal cells in intimate lateral association with the presynaptic ribbon complex were labeled. No labeling was found at the photoreceptor membrane and there was no septalaminar structure, indicative of gap junctions, between photoreceptors and horizontal cells. The focal location of Cx26 at the membrane of the dendritic tips of horizontal cells and the lack of gap junctional morphology suggests that Cx26 might form hemichannels.

Amino Acid Sequence↗

Modulation of horizontal cell function by GABA(A) and GABA(C) receptors in dark- and light-adapted tiger salamander retina.

The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark-and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABA(C) receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABA(A) receptors, and the other half is mediated by GABA(C) receptors. These results suggest that GABA released from HCs through the NO-711-sensitive GABA transporters activates GABA(A) and GABA(C) receptors, resulting in chloride conductance increase which leads to a HC depolarization and reduction of the light response. Additionally, GABA transporters also mediate GABA release in background light that is responsible for the recovery of HC membrane potential and light responses.

Adaptation, Ocular↗

An immunohistochemical study of the ontogeny of the horizontal cell in the rat retina using an antiserum against spot 35 protein, a novel Purkinje cell-specific protein, as a marker.

A sequence of morphological changes during the differentiation of retinal horizontal cells was studied by means of immunohistochemistry for spot 35 protein, a novel Purkinje cell-specific protein, in the rat retina. Spot 35-immunoreactive horizontal cell anlagen were first identified at embryonic day 18 (E18) in the outer zone of the neuroblastic cell layer as fusiform cells with slender inner and outer processes. As the development proceeded, the immunoreactive cells increased in number and intensity of the immunostaining, and the outer processes of the immunoreactive cells were often bifurcated close to their origin or at their periphery. At postnatal days 3 and 5 (P3 and P5), a regional difference in morphology of the immunoreactive horizontal cell anlagen was recognized: The immunoreactive cells near the ora serrata still assumed a bipolar shape, and those in the central portion of the retina had multiple processes. At P7 and P10, the immunoreactive cells in the periphery of the retina extended most of their outer processes in the plane of the retina, forming a network together with processes of adjacent immunoreactive horizontal cells. Their internally extending processes during these postnatal stages tended to be present in small numbers and to be short. At P14 all the immunoreactive horizontal cells extended their processes horizontally to form a tight bundle all the way along the outer plexiform layer. These findings indicate that spot 35 protein is a specific marker for the horizontal cells of the rat retina in the developmental process from their bipolar stage at E18 until reaching their mature configuration at P14.

Animals↗

Rod and cone input to horizontal cells in the rabbit retina.

In the rabbit retina, there are two types of horizontal cell (HC). The A-type HC is axonless and extensively coupled. The B-type HC is axon bearing; the somatic dendrites are radially symmetric and form a second coupled network, while the axon branches expansively to form a complex terminal structure. The B-type axon terminals (ATs) are independently coupled to form a third network in the outer plexiform layer. We have modified our dye-injection methods to obtain detailed fills of the three different horizontal cell networks for analysis via confocal microscopy. We have confirmed that A-type HCs and the somatic dendrites of B-type HCs receive input exclusively from cones, whereas the B-type ATs receive input only from rods. A single B-type AT may receive input from as many as 1,000 rods, but, surprisingly, our data reveal only one end terminal per rod spherule. The somatic dendrites of A- and B-type HCs form clusters at each cone pedicle coincident with GluR2/3 and GluR4 glutamate receptor subunits. The B-type ATs have GluR2/3- or GluR4-labeled glutamate receptors in two locations: small puncta on the end terminals within the rod spherule invagination and large clusters on the terminal stalks, approximately 1.5 microm from the rod synaptic ribbon. We conclude that AMPA receptors of the same or similar composition mediate photoreceptor input to all types of HCs.

Animals↗