Electron microscopic study of synaptic contacts between photoreceptors and HRP-filled horizontal cells in the turtle retina

The synaptic contacts between photoreceptors and horizontal cells in the retina of the turtle (Geoclemys) were studied. Horizontal cells were classified into three types according to their intracellularly recorded spectral responses: luminosity, biphasic chromaticity, and triphasic chromaticity horizontal cells (LHC, BHC, and THC). These cells were then iontophoretically filled with horseradish peroxidase (HRP). The various types of photoreceptors located within the dendritic field of the HRP-filled horizontal cells were identified either as rods or as one of the three chromatic types of cones; the latter were identified by the presence and colors of their oil droplets in the inner segments. The synaptic contacts between photoreceptors and labeled horizontal cells were then investigated by light and electron microscopy of serial sections on three LHCs, two BHCs, and one THC. LHCs made synaptic contacts with about 100 photoreceptors, including rods and three chromatic types of cones; two-thirds of these photoreceptors contacted the cell body and the remaining its axon terminal. BHCs contacted about 30 cones; red-, green-, and blue-sensitive cones in the ratio of 3:4:1. THC contacted 20 cones; red- and blue-sensitive cones in the ratio of 2:1. The dendritic processes of HRP-filled horizontal cells were found as the lateral element of the ribbon synaptic complexes. The present finding suggests that the responses of BHC and THC to red flashes are fed directly from red-sensitive cones in addition to the feedback pathway of cone-horizontal cell connections in the previous studies.     

Morphological types and connectivity of horizontal cells found in the adult zebrafish (Danio rerio) retina

We describe here different types of horizontal cells in the zebrafish retina and how they connect to photoreceptors. To label horizontal cells, crystals of DiI were placed onto the tips of pulled glass pipettes and inserted into the inner nuclear layer of fixed whole-mount retinas. The DiI-labeled horizontal cells were imaged by confocal microscopy and analyzed according to dendritic arborization, cell depth, dendritic terminal morphology, and connectivity with photoreceptors. Three types of horizontal cells were unequivocally identified: two cone-connecting (H1/2 and H3) and one rod-related cell. H1/2 cells have dendritic terminals that are arranged in "rosette" clusters and that connect to cone photoreceptors without any apparent specificity. H3 cells are larger and have dendritic terminal doublets arranged in a rectilinear pattern. This pattern corresponds to the mosaic of the single cones in the zebrafish photoreceptor mosaic and indicates that H3 cells connect specifically to either the blue-sensitive (long-single) or ultraviolet-sensitive (short-single) cones. Thus, H3 cells are likely to be chromaticity-type cells that process specific color information, whereas H1/2 cells are probably luminosity-type cells that process luminance information. Rod horizontal cells were identified by their shape and dendritic pattern, and they connect with numerous rod photoreceptors via small spherical terminals.     

Chemical synapses between turtle photoreceptors

Rod photoreceptors of the snapping turtle retina were Golgi impregnated and studied in the electron microscope. Telodendria arising from the synaptic bases ended at rods and cones as lateral or central elements of the ribbon synaptic complex, thus providing clear evidence of chemical synapses between turtle photoreceptors.     

The spatial relationship between Müller cell processes and the photoreceptor output synapse.

Glutamate is the neurotransmitter released by photoreceptors in the retina. The postsynaptic action of glutamate is terminated partly by uptake into glial (Müller) cells. The anatomical distribution of Müller cell processes around the synaptic terminals of photoreceptors was investigated electron microscopically in the tiger salamander retina. Müller cells wrap around the synaptic terminals of both rods and cones and come within 1-3 microns of the sites of glutamate release, close enough to contribute to terminating the synaptic action of glutamate.     

Decrease of gap junction permeability induced by dopamine and cyclic adenosine 3':5'-monophosphate in horizontal cells of turtle retina

The axon terminals of the H1 horizontal cells of the turtle retina are electrically coupled by extensive gap junctions. Dopamine (10 nM to 10 microM) induces a narrowing of the receptive field profile of the H1 horizontal cell axon terminals, increases the coupling resistance between them, and decreases the diffusion of the dye Lucifer Yellow in the network formed by the coupled axon terminals. These actions of dopamine involve the activation of D1 receptors located on the membrane of the H1 horizontal cell axon terminals proper. Increases of the intracellular cyclic AMP concentration induced by either stimulating the adenylate cyclase activity with forskolin or inhibiting the phosphodiesterase activity with isobutylmethylxanthine, theophylline, aminophylline, or compound RO 20-1724 elicit effects similar to those of dopamine on the receptive field profile of the H1 horizontal cell axon terminals, on their coupling resistance, and on the diffusion of Lucifer Yellow in the axon terminal network. It is concluded that dopamine decreases the permeability of the gap junctions between the axon terminals of the H1 horizontal cells of the turtle retina and that this action probably involves cyclic AMP as a second messenger.     

GABAC receptors are localized with microtubule-associated protein 1B in mammalian cone photoreceptors.

Protein MAP1B was recently reported to link GABA(C) receptors to the cytoskeleton at neuronal synapses. This interaction was demonstrated in the mammalian retina, where GABA(C) receptors were thought to be exclusively expressed in bipolar cells. Our previous studies on cultured photoreceptors suggested however the presence of GABA(C) receptors in cones. To further investigate GABA(C) receptor expression in cones, we measured GABA responses in mammalian photoreceptors in situ, and we examined the distribution of the receptor and that of protein MAP1B in the mammalian outer retina. Photoreceptors were recorded from flat-mounted retinas of retinal degeneration mice at an age when the retina becomes cone-dominated after rod cell death. GABA(A) and GABA(C)-gated currents were produced only in cones but not rods. Recording freshly dissociated retinal cells from wild-type C57 mice confirmed the presence of GABA(A) and GABA(C) receptors in cones. Immunohistochemical labeling of mouse and rat retinal sections localized GABA(C) receptors to cone terminals that were identified by peanut agglutinin lectin staining. As expected from previous studies on bipolar cells, the punctate immunostaining was not restricted to cone terminals in the outer plexiform layer. MAP1B immunolabeling was obtained in rat and pig retinas and was similarly found in cone terminals identified by the peanut agglutinin lectin staining. These results provide physiological and histological evidence that cones receive a GABA feedback in the mammalian retina and are consistent with the notion that protein MAP1B links GABA(C) receptors to the cytoskeleton at postsynaptic sites.     

Telodendrial contact of HRP-filled photoreceptors in the turtle retina: pathways of photoreceptor coupling

Synaptic contacts of photoreceptors in the turtle retina were studied by intracellular injection of horseradish peroxidase (HRP) and electron microscopy. Both cone and rod photoreceptors radiated basal processes (telodendria) from their terminal endings. These telodendria ran laterally in the outer plexiform layer. The telodendria of cones gave rise to many fine branches that penetrated synaptic cavities of several neighboring cones. Tips of these branches terminated near the walls of synaptic cavities. Some of the telodendrial contact formed two types of basal junction: symmetrical and punctate. The distribution of cones that made telodendrial contacts with the HRP-filled cone were quantitatively investigated. Green-sensitive cones (n = 3) made telodendrial contacts with neighboring red- and blue-sensitive cones, blue-sensitive cones (n = 4) with red- and green-sensitive cones, and red-sensitive cones (n = 9) with red- and green-sensitive cones. In contrast to these cone connections, rod telodendria did not penetrate neighboring photoreceptors. Direct synaptic contacts were not found between rods and cones. Our results clarify the variety of cone couplings in turtle retina: the three chromatic classes of cones are selectively coupled by the basal junctions at the ends of telodendrial processes.     

Specificity of the horizontal cell‐photoreceptor connections in the zebrafish (Danio rerio) retina

Horizontal cells (HCs) are involved in establishing the center‐surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)‐ and ultraviolet (UV)‐sensitive single cones alternate with rows of red (R)‐ and green (G)‐sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small‐field (H1 and H2) and two large‐field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1‐GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI‐labeled cone HC and connected to 50–200 rods. J. Comp. Neurol. 516:442–453, 2009. © 2009 Wiley‐Liss, Inc.     

Immunolocalization of basic fibroblast growth factor and its receptor in adult goldfish retina.

The neural retina of teleost fish can regenerate following surgical or neurotoxic lesions. As a first attempt to uncover the factors important for the regenerative response, we used immunocytochemistry to demonstrate the presence of basic fibroblast growth factor (bFGF) and its receptor in the goldfish retina. The bFGF-immunoreactivity was present throughout the retina, but was most intense in photoreceptor cells, especially cones, and Müller glia. Immunoreactivity for the bFGF receptor was strongest in the axon terminals of photoreceptors, both rods and cones. This pattern of immunolocalization is especially interesting since the proliferating cells that are thought to be responsible for generating the neural regenerate are located among the photoreceptor axon terminals. These proliferating cells have been identified as rod precursors because in the intact retina they give rise only to rod photoreceptors. When the neural retina is damaged, however, rod precursors are thought to be the source of proliferating neuroepithelial cells responsible for generating the retinal regenerate. The role played by bFGF in normal neurogenesis, cell differentiation, and/or neuronal regeneration in the fish retina has yet to be determined.     

Horizontal cells of the pigeon retina

Two types of horizontal cells are seen in Golgi-impregnated retinas of the pigeon. Type I horizontal cells are compact, “brush-shaped,” and have an axon ending as an irregular spinous arborization. The majority of the dendrites terminate in the distal part of the outer plexiform layer (OPL) as clusters which contact cones, but some terminate as single expansions in the proximal part of the OPL. The axon terminal spines are found only in the distal part of the OPL and contact both rods and cones. Pigeon Type I horizontal cells are Caja's “brush-shaped” cells, and their axon terminals resemble Caja's “stellate” cells. Type II horizontal cells have irregular, wavy, multi-branched dendrites, appear horizontally flattened, and lack axons. The dendrites terminate in the proximal part of the OPL as isolated spines and contact only cones. The Type II horizontal cells of the pigeon have not been previously described in the avian retina.     

Carrier-mediated release of GABA from retinal horizontal cells

H1 horizontal cells in goldfish retina are probably GABAergic and receive excitatory synaptic input from red cones. This input should affect the synaptic release of GABA from H1 cells. We studied the uptake and release of [3H]GABA from the isolated goldfish retina by use of autoradiography. When retinas were incubated in the light for 15 min in 0.72 microM [3H]GABA, heavy label was found over the somata (HS) and axon terminals (HAT) of H1 horizontal cells, and over pyriform amacrine cell bodies and their processes in sublamina b of the IPL. Postincubation of retinas, preloaded with [3H]GABA, in 0.5-10 mM L-glutamate or 0.1-10 mM L-aspartate, resulted in a dose-dependent and selective loss of [3H]GABA from HS and very little loss from HAT. This loss was not due to an efflux of metabolites of [3H]GABA or to any calcium-dependent vesicular release of [3H]GABA from HS. The glutamate-evoked release of [3H]GABA by H1 cells was sodium dependent, sensitive to substitution of lithium for sodium, and inhibited by nipecotic acid. In addition, [3H]GABA was released from HS by 0.1 mM ouabain but not by 50 mM potassium chloride. Our results suggest that the chemically evoked release of [3H]GABA from HS is mediated by a sodium-dependent transport carrier which may be responsible for the high affinity uptake of [3H]GABA by H1 cells as well. Since synaptic vesicles are not found at presumed synaptic release sites in H1 cells, we suggest that the GABA which is released synaptically from H1 cells may derive from a cytoplasmic pool of GABA and is released by means of a transport carrier. This carrier appears to depend primarily on the sodium concentration gradient across the H1 cell membrane rather than on the membrane potential of the H1 cell for its action. The relevance of the carrier-mediated release of GABA from HS in regard to the synaptic function of H1 cells is discussed.     

Structurally specialized contacts between the photoreceptors of the retina of the axolotl

Electron microscopic examination of the retina of the axolotl, Ambystoma mexicanum, revealed several types of morphological junctions between individual photoreceptors. Distal to the outer limiting membrane of the retina, gap junctions were found between neighboring rods and between neighboring rods and cones. In the external plexiform layer, the synaptic terminals of cones and double cones are often in positions opposite the synaptic ribbons of rod terminals that have been traditionally defined as postsynaptic. A few tight junctions between photoreceptor telodendria were observed. Relationships between the members of double cones are also described.     

Electrical coupling, receptive fields, and relative rod/cone inputs of horizontal cells in the tiger salamander retina

Light responses, dendritic/axonal morphology, receptive field diameters, patterns of dye coupling, and relative rod/cone inputs of various types of horizontal cells (HCs) were studied using intracellular recording and Lucifer yellow/neurobiotin dye injection methods in the flatmount tiger salamander retina. Three physiologically and morphologically distinct types of HC entities were identified. 1) The A-type HCs are somas that do not bear axons, with average (+/-SE) soma diameters of 20.01 +/- 0.59 microm, relatively sparse and thick dendrites, and they resemble the A-type HC in mammals. The average receptive field diameter of these cells is 529.6 +/- 10.87 microm and they receive inputs predominantly from cones. 2) The B-type HCs are broad-field somas that bear thin and long axons, with average soma diameters of 17.67 +/- 0.38 microm, thinner dendrites of higher density, and they resemble the B-type HC in mammals. The average receptive field diameter of these cells is 1,633.55 +/- 37.34 microm and they receive mixed inputs from rods and cones. 3) The B-type HC axon terminals are broad-field, coarse axon terminal processes and they resemble the B-type HC axon terminal in rabbits. The average receptive field diameter of these axon terminals is 1,291.67 +/- 24.02 microm and they receive mixed inputs from rods and cones. All these types of HC are dye-coupled with adjacent HCs of the same type. Additionally, B-type HCs and axon terminals are dye-coupled with subpopulations of bipolar cells whose axon terminals ramify in the proximal half of the inner plexiform layer, raising the possibility that these HCs may send feedforward antagonistic surround responses to depolarizing bipolar cells through electrical synapses.     

Effects of GABA and glycine on the distal cells of the cyprinid retina

Two putative retinal neurotransmitters, γ-aminobutyric acid (GABA) and glycine, were applied to the isolated carp and goldfish retinas while intracellular recordings from horizontal cells and cones were made. At relatively low concentrations (0.1–1 mM), GABA consistently hyperpolarized dark-adapted H₁ or L-type cone horizontal cells and cone photoreceptors, reduced light evoked responses and suppressed the on-transients. The effects of GABA on H₁ horizontal cells were abolished when Co²⁺ was applied to the retina, indicating that GABA exerts its effects on the horizontal cells via the receptors. Thus GABA is likely to be involved in the feedback synapse between H₁ horizontal cells and the cones. Low concentrations of glycine (0.1–1 mM), hyperpolarized a number of H₂ or C-type horizontal cells and selectively abolished their dopolarizing responses to red light; thus glycine may be involved in synaptic pathways which mediate or modulate the depolarizing responses to red flashes in C-type horizontal cells.     

Co-localization of carnosine and glutamate in photoreceptors and bipolar cells of the frog retina

Immunocytochemical methods were used to visualize carnosine (β-alanyl- -histidine)-like immunoreactivity (-LI) in the frog retina and to compare its localization with that of glutamate. Carnosine-LI was conspicuous in photoreceptors and bipolar cells. The axon terminals of labelled bipolar cells formed five bands in the inner plexiform layer. A few presumed amacrine and ganglion cells, as well as Müller cell endfeet, were also labelled. Post-embedding immunocytochemistry revealed particularly high levels of glutamate-LI in the synaptic axon terminals of bipolar cells, with a mean gold particle density 5 × higher than that of amacrine cells. Photoreceptor terminals were also labelled, but with a labelling intensity about half that of bipolar cells. Labelling of serial semithin sections showed co-localization of carnosine and glutamate in photoreceptors and bipolar cells. These findings are consistent with the notion that glutamate is the neurotransmitter of neuronal elements that transfer information vertically through the retina. We propose that carnosine may modulate GABA and/or glutamate receptors by virtue of its ability to chelate Zn²⁺ and other ions.     

Synaptic plasticity in the rod terminals after partial photoreceptor cell loss in the heterozygous rds mutant mouse.

In the retina of mice heterozygous for the retinal degeneration slow gene (rds/+) the photoreceptor cells, both rods and cones, develop abnormal outer segments but establish normal synaptic contacts. The other retinal layers also show normal structural organization. Starting from the age of 2 months, a very slow loss of photoreceptor cells progresses throughout life. As a result, the photoreceptor cell population in the retina of the affected mice is reduced to less than half at the age of 9-18 months. In some of the surviving rod terminals during this period, an increase in the number of synaptic ribbons is recorded. At the same time, the profiles of processes originating from the second order neurons and participating in these synapses are also increased in number so that the multiple ribbons appear as centres of multiple synaptic sites. Morphometric measurements of the perimeter of the synaptic profiles in rod terminals show a significant increase in the rds/+ retina over that of the control retina. Observations based on serial electron microscopy indicate that multiple synaptic sites are developed while the number of the second order neuronal processes, entering the terminals, remains unchanged. The frequency of terminals with multiple synapses in the rds/+ retina increases with progressive photoreceptor cell loss. Similar changes do not occur in the terminals of the cones. It is postulated that loss of some rod photoreceptor cells within a group that is presynaptic to common bipolars or horizontal cells results in partial deafferentation which in turn stimulates the growth of the remaining synaptic elements. The possible compensatory effect and functional significance of such synaptic growth are discussed.     

Identification of a cone bipolar cell in cat retina which has input from both rod and cone photoreceptors

It has been generally accepted that rod photoreceptor cells in the mammalian retina make synaptic contact with only a single population of rod bipolar cells, whereas cone photoreceptors contact a variety of cone bipolar cells. This assumption has been challenged in rodents by reports of a type of cone bipolar cell which receives input from both rods and cones. Questions remained as to whether similar pathways are present in other mammals. We have used an antiserum against the glutamate transporter GLT1-B to visualize a population of cone bipolar cells in the cat retina which make flat contacts with axon terminals of both rod and cone photoreceptor cells. These cells are identified as OFF-cone bipolar cells and correspond morphologically to type cb1 (CBa2) cone bipolar cells which are a major source of input to OFF-beta ganglion cells in the cat retina. The GLT1-B transporter was also localized to processes making flat contacts with photoreceptor terminals in rat and rabbit retinas. Examination of tissue processed for the GluR1 glutamate receptor subunit showed that cb1 cone bipolar cells, like their rodent counterparts, express this alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-selective receptor at their contacts with rod spherules. Thus, a direct excitatory pathway from rod photoreceptors to OFF-cone bipolar cells appears to be a common feature of mammalian retinas.     

Catecholaminergic, GABAergic, and hippocamposeptal innervation of GABAergic "somatospiny" neurons in the rat lateral septal area

This study deals with the neurochemical characterization of the rat lateral septal area (LSA) somatospiny neurons and their innervation by hippocamposeptal, catecholaminergic, and GABAergic fibers. Electron microscopic single and double immunostaining methods were used to label catecholaminergic fibers and GABAergic cells and boutons. Axon terminals originating in the hippocampus were labeled by acute anterograde axon degeneration induced by fimbria-fornix transection 36 hours before sacrifice. Three types of experiments were performed. The convergent catecholaminergic and hippocamposeptal innervation of LSA somatospiny neurons was studied by combining immunostaining for tyrosine hydroxylase (TH) with fimbria-fornix transection. GABAergic neurons and their hippocamposeptal afferents were identified and characterized in colchicine pretreated animals immunostained for glutamic acid decarboxylase (GAD) combined with fimbria-fornix transection. The third experiment aimed at simultaneously visualizing the relationships between catecholaminergic boutons, hippocamposeptal excitatory amino acid containing axon terminals and GABAergic profiles by double immunostaining for TH (the PAP technique) and GAD (the immunogold method) combined with fimbria-fornix transection. The results are summarized as follows: 1) The same LSA somatospiny neurons receive synaptic inputs from the hippocampus and TH immunoreactive fibers which form pericellular baskets around these cells. 2) LSA somatospiny neurons are GABAergic and are postsynaptic targets of GABAergic boutons with unknown origin and hippocamposeptal axon terminals. 3) The double immunostaining experiment, finally, provided direct evidence that the same GABAergic somatospiny neurons are postsynaptic targets of both catecholaminergic and hippocamposeptal afferents. The synaptic interconnections described in this study provide anatomical basis for a better understanding of the action of catecholamines, excitatory amino acids, and GABA on the activity of LSA neurons.     

Immunocytochemical analysis of GABA-positive and calretinin-positive horizontal cells in the tiger salamander retina

By using immunocytochemical techniques, we demonstrate that there are two distinct, nonoverlapping populations of horizontal cells (HCs) in the tiger salamander retina: GABA-positive cells account for about 72% and GABA-negative (calretinin-positive) cells account for 28% of the total HC somas. The calretinin-positive HCs have relatively sparse and thick dendrites: soma diameter of 19.72 +/- 0.29 microm, and soma density of 140 +/- 13 cells/mm(2), morphological features very much like the A-type HCs described in the accompanying article. The GABA-positive HCs have thinner dendritic and coarse axon-terminal-like processes of higher density: soma diameter of 18 +/- 0.18 microm, and soma density of 364 +/- 18 cells/mm(2), features that very much resemble the B-type HCs and B-type HC axon terminals in the accompanying article. By using double and triple immunostaining techniques we found that only 18% of the non-GABAergic HC dendritic clusters contact rods, whereas the remaining 82% of the dendritic clusters contact cones. This is consistent with the physiological finding in the accompanying article that the A-type HCs are cone-dominated. On the other hand, 32% of GABAergic HC dendrites contact rod pedicles and 68% contact cone pedicles, consistent with the physiological finding that B-type HCs and B-type HC axon terminals receive mixed rod/cone inputs. Detailed confocal microscope analysis shows that 4% rods, 6% principal double cones/single cones, and 100% accessory double cones contact calretinin-positive HCs, and 79% rods, 100% principal double cones, 14% accessory double cones, and 82% single cones contact GABAergic HCs. These results suggest that GABAergic and non-GABAergic HC input/output synapses differ and they may mediate different functional pathways in the outer retina.