Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

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Figure 2 Bipolar cells and their cone contacts in the primate and mouse retina. (a, b) Horizontal view of Golgi-impregnated midget bipolar cells, with the plane of focus at their dendritic tips in the OPL. The micrographs show examples of a single-cone contacting (a) and a two-cone-contacting (b) midget bipolar cell.

(c) Horizontal view of a Clomeleon mouse retina (Clm1 line) double labeled for GFP (green) and for GluR5 (red). The clusters of GluR5 puncta represent individual cone pedicles. The dendrites of the blue cone bipolar cell (asterisk) contact threecone pedicles (circles) and avoid all other pedicles. The BB cell is cone-selective for S-opsin expressing cones. (d, e) Dendritic trees of two type 7 bipolar cells in the Gus-GFP mouse and their cone contacts (circles). Type 7 cells contact on average 8.4 cones. Scale bars ¼10 mm. (a, b) Images: courtesy of H. Wa¨ssle. From Puller, C., Haverkamp, S., and Gru¨nert, U. (2007).

OFF midget bipolar cells in the retina of the marmoset,

Callithrix jacchus, express AMPA receptors. Journal of Comparative Neurology 502: 442–454.

pigments, it has been estimated that the L/M separation in the old world primate lineage occurred 35 million years ago. The separation of the M- and S-cone pigments occurred >500 million years ago and thus represents the phylogenetically ancient, primordial color system. The morphological substrate for the dichromatic color vision common to most placental mammals is the S-cone pathway. Bipolar cells selective for S-cones in the macaque monkey retina have long, smoothly curved dendrites and contact between one and three cone pedicles. Their axons terminate in rather large varicosities in the innermost part

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Figure 3 Bipolar cell contacts at photoreceptor terminals. (a, b) Schematic drawings showing the arrangement of contacts at a cone pedicle (a) and rod spherule (b). Horizontal cell processes

(H) always end laterally and deeper in the invaginations of both rod and cone terminals. In cone pedicles, the central processes derive from invaginating ON bipolar cells (IB), whereas in rod spherules, the central elements derive from rod bipolar cells (RB). Flat contacts at the base of the cone pedicle are subdivided into triad associated (TA) or nontriad associated (NTA) contacts, depending on their relative distance from the triad. Some cone bipolar cells receive direct input from rods (CB in (b)). (c, d) Type 4 cone bipolar cells of the mouse retina express calsenilin and contact cones as well as rods. Preembedding electron micrographs showing several flat contacts of a calsenilin-positive dendrite at a cone pedicle base (arrows in (c)) and a calsenilinpositive dendrite making a flat contact on a rod spherule

(arrow in (d)). Scale bar ¼0.5 mm. (a, b) Schematic drawings: courtesy of C. Puller.

of the IPL, close to the ganglion cell layer (BB cells in Figure 1(b)) and innervate the inner tier of the dendritic tree of the small bistratified ganglion cells: These cells are color-opponent and respond to increasing blue light (blue – ON) and decreasing yellow light (yellow – OFF).

Immunostaining with antisera specific for S-opsin has shown that S-cones constitute approximately 10% of the cones in most mammalian retinas. However, in some species S-cones have a very uneven topographical distribution across the retina and many cones express both M- and S-opsin. Recently, a transgenic mouse line could be studied, where Clomeleon, a genetically encoded fluorescence indicator, was expressed under the thy1 promoter. Clomeleon was expressed in ganglion cells, amacrine cells, and bipolar cells.

Clomeleon-labeled ganglion cells, amacrine cells and bipolar cells

Among the bipolar cells the S-cone-selective (blue cone) type could be identified, and the cone-selective contacts

and the retinal distribution could be studied (Figure 2(c)). The morphological details of the blue cone bipolar cells match type 9 cells of mice (Figure 1(a)) and they are closely similar to the blue cone bipolar cell of the primate retina. It is interesting that in the ventral mouse retina, where most cones express both M- and S-opsin, blue cone bipolar cells contact only those cones, which express S-opsin only. They are the genuine S-cones of the mouse retina. Meanwhile, S-cone-selective bipolar cells have also been verified in ground squirrel and rabbit retina.

Diffuse Bipolar Cells

Most bipolar cell types of the mammalian retina contact between 5 and 10 neighboring cones (Figure 2(d) and 2(e)). In mouse, the number of cone pedicles contacted by individual bipolar cells varied from an average of 5.6 for type 2 cells to an average of 8.4 for type 7 cells. Each and all cones are contacted by at least one member of any given type of bipolar cell (leaving aside the blue cone pathway). Consequently, each cone pedicle is connected to a minimum of eight different bipolar cells. They represent eight separate channels that transfer the light signal into the IPL. Parallel processing, therefore, starts at the first synapse of the retina, the cone pedicle.

Diffuse bipolar cells of the primate retina contact L- and M-cones in their dendritic field non-selectively. Whether all diffuse bipolar cell types also contact S-cones is still a matter of discussion, and it has been proposed that one type of diffuse bipolar cell avoids S-cones in the primate retina. This type would be a good candidate to transfer a yellow (L-plus M-cone) signal into the IPL, where it could contact the outer tier of the dendritic tree of the small bistratified ganglion cells. For the dichromatic ground squirrel retina, it has been shown that diffuse bipolar cells sample cone signals differently: some types receive mixed S/M-cone input and other types receive an almost pure M-cone signal. Bipolar cells that sum signals from S- to M-cones are therefore involved with the transfer of luminosity signals, whereas bipolar cells that carry M-cone signals can have, together with S-cone bipolar cells, a role in color discrimination. Alternatively, these bipolar cells may mediate – due to their relatively small dendritic fields – high acuity vision. This idea would correspond to the fact that the center of the human fovea, which mediates the highest acuity vision, also excludes S-cones.

Like the midget bipolar cells, diffuse bipolar cells also differ in their synaptic contacts with cone pedicles, making either flat or invaginating contacts. EM reconstructions of Golgi-impregnated diffuse bipolar cells of the macaque monkey retina revealed that DB1, DB2, and DB3, which have their axon terminals in the outer IPL and are putative OFF bipolar cells (Figure 1(b)), make exclusively basal junctions with the cone pedicle.

They always have triad associated (TA) and nontriad associated (NTA) contacts (Figure 3(a)). The proportion of TA and NTA contacts varies according to the cell type, as does the average number of contacts per cone, which is between 10 and 20. Bipolar cells DB4, DB5, and DB6 have their axon terminals in the inner part of the IPL and are putative ON bipolar cells. They have an average of between four and eight invaginating synapses per cone pedicle. In addition, they also form basal junctions, in a predominantly TA position. Thus, while the dichotomy ‘invaginating ¼ON, flat ¼OFF’ holds for midget bipolar cells, it does not conform so clearly for diffuse bipolar cells. Therefore, the type of synapse made by a bipolar cell at a cone pedicle, flat versus invaginating, is not the decisive feature; it is rather the glutamate receptor expressed there.

Cone Bipolar Cells with Rod Input

Recent results from rodent and rabbit retina have shown that some OFF cone bipolar cells make also basal contacts with rod spherules and thus receive a direct input from rods. This represents a third route for the rod signal, in addition to the rod bipolar cell circuit, and the gap junctions between rods and cones. In mouse, true-cone- selective OFF bipolar cells (types 1 and 2, Figure 1(a)) can be distinguished from types with mixed rod-cone input (types 3 and 4, Figure 1(a)). Type 4 bipolar cells make several basal contacts at the cone pedicle base (Figure 3(c)) and an individual cell contacts five to eight cones. In addition, some dendrites extend further out into the OPL and contact rod spherules as flat contacts (Figure 3(d)). On average, we counted 10 rod spherule contacts per type 4 bipolar cell, and approximately 10% of rods contacted by type 4 bipolar cells.

Immunocytochemical Markers and

Transgenic Mouse Lines

The morphological classification of bipolar cells has been made more objective and more quantitative by immunocytochemical markers that selectively label specific cell types. Some of the markers label the same cell types across different species. For instance, rod bipolar cells of all mammals are immunoreactive for protein kinase C a (PKCa). However, other markers, such as calcium-binding proteins, label different cell types in different mammals. Calbindin antibodies label the DB3 OFF cone bipolar cell in the primate retina. However, in the rabbit retina, the calbindin-immunoreactive cell is an ON cone bipolar cell, and in the rat and mouse retina, no bipolar cell expresses calbindin immunoreactivity. Recoverin is another example of a marker that selects different types of bipolar cells in different species. While

in rat and rabbit retinas two types of bipolar cell, an OFF and an ON cone bipolar cell, appear to be labeled, only the OFF midget bipolar cell is labeled in the macaque monkey retina. Even in closely related species, different bipolar cell types can be selected by the same marker. The antibody against the carbohydrate epitope CD15 labels a single population of ON bipolar cells (DB6) in macaque monkey, whereas DB6 cells and OFF midget bipolar cells are labeled in marmoset monkeys. In rabbit, CD15 antibodies label an ON cone bipolar cell, whereas in mouse CD15 is expressed in OFF cone bipolar cells. In contrast, antibodies against the calcium-binding protein CaB5 immunolabel at least three types of bipolar cells in a variety of mammalian species. In all species, rod bipolar cells, one ON cone bipolar cell and at least one OFF cone bipolar cell were labeled (Figure 4).

The IPL can be subdivided into five strata of equal thickness (Figures 1 and 5). In mouse, these strata can be easily defined by immunolabeling the retina for the calcium-binding protein calretinin (Figure 5(b)), which reveals three densely labeled horizontal bands of processes. The outer band (between stratum 1 and 2) contains the processes of the OFF cholinergic amacrine cells and the outer dendritic branches of direction-selective ganglion cells. The band in the inner IPL (between stratum 3 and

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OFF-CB

ON-CB

RB

Mouse

(b)

Cat

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Monkey

Figure 4 Comparison of CaB5 immunoreactivity in mouse (a), cat (b), and monkey (c) retina. In all three species, rod bipolar cells (RB) and at least one ON cone bipolar (ON-CB) and one OFF cone bipolar cell (OFF-CB) are labeled. Scale bar ¼50 mm.

4) contains the processes of the ON cholinergic amacrine cells and the inner dendritic branches of direction-selective ganglion cells. The band in the middle of the IPL (between stratum 2 and 3) represents the level of stratification of a nitric oxide synthase (NOS) immunoreactive amacrine cell type and separates the OFF sublamina (outer) from the ON sublamina (inner). The calcium-binding protein 5 (CaB5)- immunoreactive bipolar cells stratify in three strata; in stratum 2 where the type 3 bipolar cells stratify, in stratum

Type 2

Type 6

Figure 5 Immunocytochemical staining of mouse bipolar cells. (a, b) Vertical section through a mouse retina that was double immunostained for CaB5 (red) and calretinin (green). Three dendritic strata within the IPL express calretinin and subdivide the IPL into four sublaminae. Three bipolar cell types (type 3, type 5, and RB) express CaB5. Their axons terminate in the IPL in sublamina 2, sublamina 3, and sublamina 5, respectively. (c, d) Vertical section through the Gus–GFP mouse retina immunostained for GFP (green) and CaB5 (red). The retinal layers are indicated (OPL, INL, IPL, subdivided into five sublayers of equal thickness; GCL). Type 7 bipolar cells express high levels of GFP and their axons terminate at the border of sublaminae 3/4. Rod bipolar cells also express GFP, but weakly (double labeled with Cab5 in (d)). (e, f ) Vertical section through the mouse retina immunostained for synaptotagmin 2 (Syt2 , green) and CaB5 (red). Type 2 and type 6 bipolar cells express Syt2. Type 2 axons terminate in sublamina 1/2, above the CaB5-labeled type 3 axons; type 6 axons terminate mainly in sublamina 4/5 and intermingle with CaB5-labeled RB axon terminals. Scale

bar ¼25 mm.

3 where type 5 cells stratify, and in stratum 4/5 where the rod bipolar cells terminate (Figure 5(a) and 5(b)).

We have used several selective markers, either antibodies or the specific expression of fluorescent proteins in transgenic mouse lines, for analyzing the different types of bipolar cells in the mouse retina. Five putative OFF cone bipolar cells were analyzed by selective markers. Type 1 bipolar cells were found to be immunoreactive for the neurokinin 3 receptor (NK3R) and they could also be identified in Clm1 transgenic mice. Type 2 bipolar cells expressed NK3R and synaptotagmin II (Syt2) immunoreactivity (Figure 5(e) and 5(f)). Type 3a and type 3b cells were immunostained for the hyperpolarizationactivated cyclic nucleotide-gated potassium channel 4 (HCN4) and the protein kinase A regulatory subunit II b (PKARIIb), respectively, and type 4 cells expressed the calcium-binding protein, calsenilin. In the case of ON cone bipolar cells, markers for four types were described. Type 5 bipolar cells were labeled in the 5- hydroxytryptamine 3 receptor-EGFP (5HT3R-EGFP) transgenic mouse; however, they represent two types (named 5a and 5b). Type 6 bipolar cells were partially identified because their axons express Syt2 (Figure 5(e) and 5(f )). Type 7 bipolar cells were labeled in the

Gus-GFP mouse, a transgenic mouse line where GFP is expressed under the control of the gustducin promoter (Figures 2(d), 2(e), 5(c) and 5(d)) Type 9, the blue cone bipolar cell, has been identified in Clm1 mice. Rod bipolar cells have been immunostained for PKCa. This list suggests that, with the exception of type 8 bipolar cells, selective markers, which stain the whole population, are available for all bipolar cell types of the mouse retina.

Synaptic Contacts of Bipolar Cells in the

Inner Plexiform Layer

The axons of bipolar cells terminate in the IPL in lobular swellings (Figure 6(a)). Some bipolar cell types, such as DB3 and DB6 of the primate retina and type 7 of the mouse retina, keep their axon terminals within a narrow stratum (Figure 5(c) and 5(d)). Hence, their output will be restricted to the amacrine and ganglion cell dendrites they meet within that stratum. Other bipolar cells such as type 4 and type 6 of the mouse retina occupy with their axon terminals the complete OFF or ON sublamina respectively (Figure 5(e) and 5(f )). They are possibly engaged in contacts with a wider variety of postsynaptic

neurons. Midget bipolar cells of the primate retina represent a special case, because their axon terminals precisely match in width and depth the dendritic tops of midget ganglion cells, and they together form a densely interconnected glomerulus. The axon terminals of neighboring bipolar cells of a given type usually tile the retina without overlap in the horizontal direction. An interesting question is how the precisely layered and territorial arrangement of axon terminals is formed during embryonic development. Type-specific interactions have to be postulated because terminals of different types can overlap within the same sublamina. Proteins such as the Down’s syndrome cell adhesion molecule (Dscam) and an immunoglobulin superfamily protein (Sidekick) are involved in laminaspecific segregations of neuronal processes within the IPL during embryonic development.

Bipolar cell axon terminals provide synaptic output through multiple ribbon synapses (Figure 6(c)). We have counted the number of output synapses of the type 7 bipolar cells in the Gus–GFP mouse. The numbers varied between 74 and 128 (n ¼ 8) depending on the size of the axon terminals. The number of ribbon synapses made by rod bipolar cells of the rabbit retina was up to 30, compared to only 15 in the rat retina, which reflects the smaller size of rod bipolar axon terminals in rats. The fine structure of the bipolar cell output synapses is shown in Figure 6(b). The presynaptic ribbon is surrounded by vesicles and two

postsynaptic elements. This synaptic arrangement is named a dyad. One of the postsynaptic partners at cone bipolar cell dyads is usually a ganglion cell dendrite, while the other one is an amacrine cell process. The amacrine cell process often makes within about 0.5–1.0 mm of the dyad a conventional synapse back onto the bipolar cell axon terminal. This arrangement appears to be a reciprocal synapse and because most amacrine cells are inhibitory, it is the structural correlate of negative feedback at the dyad. Bipolar cell axons receive, in addition to reciprocal synapses, input from amacrine cells not related to the dyads. In the case of rod bipolar cell dyads, both postsynaptic partners are amacrine cells (AI and AII); and AI cells provide the reciprocal synapses.

Costratification of Preand Postsynaptic

Partners in the Inner Plexiform Layer

Bipolar axons terminate at distinct levels within the IPL, and different types of amacrine and ganglion cells also keep their processes at specific levels within the IPL, which leads to the prediction that they are also engaged in mutual synaptic contacts (Figure 7). However, this simple rule has only been verified in a few instances. Midget bipolar cells of the primate retina – both ON and OFF midget – contact midget ganglion cells. Parasol

ganglion cells of the primate retina also occur as OFF and ON pairs and their dendrites stratify in sublamina 2 and sublamina 4, respectively. The OFF parasol cells receive their major, excitatory input from DB3 bipolar cells, and ON-parasol cells most likely from DB5 bipolar cells. In rabbit, ON a ganglion cells stratify just below the ON cholinergic amacrine cells and cofasciculate (distribute together) with the axon terminals of the calbindinimmunoreactive bipolar cells.

The bistratified ON/OFF direction-selective (DS) ganglion cell coincides with the level of stratification of ON and OFF cholinergic amacrine cells. In the rabbit retina, DS ganglion cells receive the majority of their synaptic input from amacrine cells. The axon terminals of the CD15-immunoreactive bipolar cells stratify slightly more distally of the ON cholinergic band. In addition, they follow the pattern of the ON cholinergic dendrites, and are, therefore, good candidates for providing synaptic input to the DS circuitry.

Figure 7(a) shows a double labeling of glycogen phosphorylase (glypho) and CaB5 in the macaque monkey retina. The glypho-immunoreactive cells occur – like the cholinergic amacrine cells – as mirror-symmetrical populations of regular and displaced wide-field amacrine cells. The regular amacrine cells branch in sublamina

2 and co-stratify with DB3 cells; the displaced amacrine cells branch in sublamina 3 and co-stratify with DB4 cells.

Transgenic mouse lines are extremely helpful to study potential contacts between bipolar cells and their postsynaptic partners in the mouse retina. For instance, in the GFP-O line, each mouse expresses GFP in a small and variable set of ganglion cells and small-field amacrine cells. See Figure 7(b) for a small-field amacrine cell and Figure 7(c) for a monostratified ON ganglion cell, both of which coincide with the type 5 bipolar cell.

Bipolar Cells of Nonmammalian

Vertebrates

It has been shown that bipolar cells of a particular cell are usually found in one-half of the IPL or the other, but not in both. In several nonmammalian vertebrates, however, the axon terminals of the bipolar cells are highly stratified, ending on one or several levels in the inner plexiform layer. Most cold-blooded vertebrate retinas contain large bipolar cells, long thought to be rod-related bipolar cells, and small bipolar cells, believed to be cone-related bipolar cells. In fish, it has been shown that many bipolar cells, particularly the larger ones, contact both rods and cones.

It has also been shown that certain of these bipolar cells are likely to be color-coded, because they contact specific sets of cones. In turtle retina, at least 15 different morphological types of bipolar cells were found (Figure 8(a)). Some are monostratified with only a single axon terminal (B1, B2, B5, B11, and B12), several are bistratified (B4, B6, B8, B9, and B10), and some are tristratified (B3, B7, B13, B14, and B15). All seem to have Landolt clubs arising from their dendrites in the outer plexiform layer (OPL) to extend into the outer nuclear layer. A functional organization of the turtle IPL into OFF sublaminae (strata 1 and 2) and ON sublaminae (strata 3, 4, and 5), as has been described for other vertebrate retinas, is quite clear for two types of OFF bipolar cells, which stratify in the two distal strata (B4, B5) and for all four types of ON bipolar cells (B1, B2, B6, and B7). However, some OFF bipolar cells (B3, B9, B10, and B13) have axon terminals in strata 3–5 in addition to their terminations in stratum 1 or 2 (Figure 8(a)).

Color-Coded Bipolar Cells in the Turtle Retina

The turtle has excellent color vision, and is at least tetrachromatic. Three cones are sensitive to long wavelengths, one to medium wavelengths, one to short wavelengths, and a further cone to ultraviolet light. The chromatic types of cones can be morphologically identified by the presence and colors of their oil droplets and the shape of their pedicles. Two of the bipolar cell types in turtle are color-opponent: B10 is a red-ON, green/blue-OFF bipolar cell with axons in S2 and S4 and B11 is a red-OFF, green/blue-ON bipolar cell with an axon terminal in S3 (Figure 8(a)).

We have analyzed the cone contacts of a horse-radish peroxidase labeled B10 cell by serial EM reconstruction: 45 ribbon-associated synapses were found in single and double L-cones; basal junctions were found in M-cones (Figure 8(b)–8(d)). No contacts were found with rods, S-cones, and UV-cones. The results showed that invaginating synapses with L-cones and noninvaginating synapses with M-cones formed the basis of color opponency in an identified bipolar cell for red versus green light stimulation. We suggested sign-inverting transmission from L-cones at invaginating synapses mediated by G-protein-coupled metabotropic glutamate receptors, and sign-conserving transmission from M-cones at widecleft basal junctions mediated by ionotrophic (ion-gated) glutamate receptors. For B11 bipolar cells, we would predict that the dendrites express ionotropic glutamate receptors at their contacts with L-cones (red-OFF) and

metabotropic glutamate receptors at their contacts with M-cones (green-ON).

See also: Cone Photoreceptor Cells: Soma and Synapse; Information Processing: Bipolar Cells; Morphology of Interneurons: Amacrine Cells; Morphology of Interneurons: Interplexiform Cells; Rod and Cone Photoreceptor Cells: Inner and Outer Segments; Rod Photoreceptor Cells: Soma and Synapse.

Further Reading

Ammermu¨ller, J. and Kolb, H. (1996). Functional architecture of the turtle retina. Progress in Retinal Research 15: 393–433.

Boycott, B. B. and Wa¨ssle, H. (1991). Morphological classification of bipolar cells in the macaque monkey retina. European Journal of Neuroscience 3: 1069–1088.

Boycott, B. B. and Wa¨ssle, H. (1999). Parallel processing in the mammalian retina. The Proctor Lecture. Investigative Ophthalmology and Visual Science 40: 1313–1327.

Chan, T. L., Martin, P. R., Clunas, N., and Gru¨nert, U. (2001). Bipolar cell diversity in the primate retina: Morphologic and immunocytochemical analysis of a new world monkey, the marmoset Callithrix jacchus.

Journal of Comparative Neurology 437: 219–239.

Euler, T., Schneider, H., and Wa¨ssle, H. (1996). Glutamate responses of bipolar cells in a slice preparation of the rat retina. Journal of Neuroscience 16: 2934–2944.

Famiglietti, E. V. (1981). Functional architecture of cone bipolar cells in mammalian retina. Vision Research 21: 1559–1563.

Ghosh, K. K., Bujan, S., Haverkamp, S., Feigenspan, A., and Wa¨ssle, H. (2004). Types of bipolar cells in the mouse retina. Journal of Comparative Neurology 469: 70–82.

Haverkamp, S., Mo¨ckel, W., and Ammermu¨ller, J. (1999). Different types of synapses with different spectral types of cones underlie color opponency in a bipolar cell of the turtle retina. Visual Neuroscience 16: 801–809.

Haverkamp, S., Wa¨ssle, H., Du¨bel, J., et al. (2005). The primordial, blue cone color system of the mouse retina. Journal of Neuroscience 25: 5438–5445.

Haverkamp, S., Specht, D., Majumdar, S., et al. (2008). Type 4 OFF cone bipolar cells of the mouse retina express calsenilin and contact cones as well as rods. Journal of Comparative Neurology 507: 1087–1101.

Kolb, H. (1970). Organization of the outer plexiform layer of the primate retina: Electron microscopy of Golgi-impregnated cells.

Philosophical Transactions of the Royal Society, London, B 258: 261–283.

Li, W. and DeVries, S. H. (2006). Bipolar cell pathways for color and luminance vision in a dichromatic mammalian retina. Nature Neuroscience 9: 669–675.

MacNeil, M. A., Heussy, J. K., Dacheux, R. F., Raviola, E., and Masland, R. H. (2004). The population of bipolar cells in the rabbit retina.

Journal of Comparative Neurology 472: 73–86.

Sherry, D. M. and Yazulla, S. (1993). Goldfish bipolar cells and axon terminal patterns: A Golgi study. Journal of Comparative Neurology 329: 188–200.

Wa¨ssle, H., Puller, C., Mu¨ller, F., and Haverkamp, S. (2009). Cone contacts, mosaics and territories of bipolar cells in the mouse retina.

Journal of Neuroscience 29: 106–117.

Glossary

Cone pedicle – The axonal synaptic ending of the cone photoreceptor in the outer plexiform layer

of the retina; it is commonly a relatively large conical structure that contains about 30 synaptic sites (triads), each having a presynaptic ribbon and three postsynaptic processes.

Connexin – A transmembrane protein; six connexins form a connexon or hemichannel and two connexons form a gap junction.

Connexins are a diverse family and can combine into homomeric or heteromeric connexons and gap junctions.

Ephaptic – An action mediated by an electrical contact between nerve cells, without the mediation of a neurotransmitter. In the case of horizontal cells it is mediated by a hemichannel.

Gap junction – A specialized connection between neighboring cells, forming an electrical synapse in the neurons. A gap junction consists of two connexons (hemichannels) that form an intercellular pore across the touching cell membranes. The connexons are connexin hexamers. The pore allows various ions and molecules (e.g., neurobiotin) to pass between the cells. Gap junctions can be regulated (opened and closed) by

neuromodulators and thus implement a flexible functional syncytium.

Rod spherule – The axonal synaptic ending of the rod photoreceptor in the outer plexiform layer

of the retina; it is a small globular structure that contains two synaptic sites with a presynaptic ribbon and four postsynaptic processes.

Triad – A synaptic arrangement at the synaptic ending of a photoreceptor, located at an invagination of the cone pedicle or rod

spherule. The presynaptic side of the triad contains an electron-dense synaptic ribbon for the

docking of transmitter vesicles. The postsynaptic side has three invaginating processes, a central bipolar cell dendritic terminal and two lateral horizontal cell terminals. Rod triads receive two biploar cell terminals and two horizontal cell terminals.

General Morphology and Connectivity

Basic Morphology

Horizontal cells are interneurons of the outer retina and the largest neurons present in that region. Their somata are located in the outer part of the inner nuclear layer. Their processes ramify in the outer plexiform layer and form synaptic contacts with the photoreceptors. The horizontal cells of mammals – to which this article is limited – comprise two types, commonly termed A-type and B-type; the terms A-HC or HA and B-HC or HB are also in use. The B-type has a smaller, densely branched dendritic tree with relatively fine dendrites and also has an axon ending in a profusely branched axon terminal system. The A-type has a larger, more sparsely branched dendritic tree with fewer and stouter primary dendrites, and has no axon (Figure 1(a)). The dendrites of both types carry clusters of terminals (terminal aggregates) that synapse exclusively with cones. The axon terminal system of the B-type has unclustered terminals that exclusively contact rods (Figures 1(b)–1(g)).

The horizontal cell terminals, together with the dendrites of invaginating bipolar cells (ON bipolar cells, depolarizing to the onset of light), insert into invaginations at the base of the photoreceptor terminal (cone pedicle or rod spherule). The synaptic complexes thus formed are referred to as triads. Each cone pedicle possesses a substantial number of invaginations with triads (20–50 in primates), whereas a rod spherule commonly only has one invagination with two triads (Figures 1(e)–1(g)). Presynaptically, the triad is marked by a synaptic ribbon that is thought to play a crucial role in the continuous vesicular release of the photoreceptor transmitter glutamate. Postsynaptically, each mammalian triad has a bipolar cell dendritic terminal (two at rod spherules) as a central element, flanked by two horizontal cell terminals as lateral elements. The bipolar cells, via their axonal contacts, pass their signals on to the inner retina. The horizontal cells have no spatially segregated output synapses. The only synaptic contacts they form are with the photoreceptors. These are their input and output sites, representing a local feedback mechanism for the cones (by A-type and B-type dendrites) and the rods (by B-type axon terminals).

Horizontal cells modulate signal transmission from the photoreceptor to the invaginating bipolar cell processes.

B-type

ats

A-type

50 m

(a)

Figure 1 Basic mammalian horizontal cell morphologies.

(a) Flat views of a Golgi-stained B-type horizontal cell with axon terminal system (ats) and axonless A-type horizontal cell of cat. Middle row: (b) enlarged part of the B-type ats with single terminals that are the contacts with rods; (c) a schematic photoreceptor pattern with the rather regularly spaced cones surrounded by the more numerous and smaller rods; and (d) part of an A-type dendrite with clustered terminals that are the contacts with cones. Bottom row: (e) schematic triad arrangements at a rod spherule; (f) electron micrograph of a triad in a mouse rod spherule; and (g) schematic triad arrangements at a cone pedicle. R, rod spherule; C, cone pedicle; H, horizontal cell process; B, bipolar cell process; r, presynaptic ribbon; and m, mitochondrion. For details, see text. (a) Adapted from Boycott, B.B., Peichl, L., and Wa¨ssle, H. (1978). Morphological types of horizontal cell in the retina of the domestic cat.

Proceedings of the Royal Society (London) B 203: 229–245.

(f) Image kindly provided by Silke Haverkamp. Adapted from Figure 1 in Peichl, L., Sandmann, D., and Boycott, B. B. (1998). Comparative anatomy and function of mammalian horizontal cells. In: Chalupa, L. M. and Finlay, B. L. (eds.) Development and Organization of the Retina: From Molecules to Function, pp. 147–172. New York: Plenum Press. With kind permission of Springer Science and Business Media.

The horizontal cell feedback is inhibitory and supposedly creates the antagonistic receptive field surrounds of bipolar cells, thus contributing to the receptive field properties of the ganglion cells. However, the exact nature of the feedback synapse is unknown. There is some evidence that mammalian horizontal cells use the inhibitory transmitter gamma aminobutyric acid (GABA); however,

a feedback mediated by electrical conduction (ephaptic, via hemichannels) and pH modulation (proton hypothesis) is also considered. In addition, the horizontal cells probably have feed-forward connections to ON bipolar cells. OFF bipolar cells, which make flat (noninvaginating) contacts at the photoreceptor base, are not in direct apposition to the horizontal cells. It is assumed that the horizontal cell modulation affects them indirectly via the changed photoreceptor output.

The dendritic spread of a horizontal cell is much larger than that of a cone bipolar cell, and activation by cones anywhere in its dendritic field results in an output across the entire dendritic field. Thus, information about the illumination state of cones outside a bipolar cell’s input region is picked up by the overlying horizontal cells and negatively fed back to the few cones providing the direct bipolar cell input – the classical lateral inhibition that sharpens contrast sensitivity and acuity. Most likely, the same function is provided for the rod pathway by the B-type axon terminal system.

Photoreceptor Contacts

In the mammalian retina, the rod and cone signals are separately carried by cone and rod bipolar cells, respectively. These major rod and cone pathways only converge in the inner retina. In addition, there is gap-junctional coupling between rods and cones, and a few cone bipolar cells also synapse with rods. Mammalian horizontal cells separately serve the rod and cone pathways.

Contacts with cones

The dendritic fields of horizontal cells are commonly shaped round to oval. Depending on the retinal location, A-type dendritic trees in cat are 80–220 mm in diameter and contact 120–170 cones, whereas B-type dendritic trees are 70–120 mm in diameter and contact 60–90 cones. A- and B-type cells in rabbit are slightly larger but contact similar numbers of cones. In their cone connections, the two horizontal cell types share a common input. Most mammals are cone dichromats with an approximately 90% majority of medium-to-long-wavelength-sensitive (L) cones and a 10% minority of short-wavelength-sensitive

(S) cones. In most studied species, both A- and B-type cells contact the vast majority of cones within their dendritic fields; this was considered as evidence that each spectral cone type contacted each horizontal cell type. Soma recordings from cat and rabbit horizontal cells with spectral stimuli confirmed that both types hyperpolarize to all wavelengths. Direct anatomical evidence for the nonselective contacts of both horizontal cell types with both spectral cone types has been obtained in rabbit and tree shrew (Figures 2 and 3). In cone triads, the two lateral elements are either two A-type terminals, two B-type terminals, or one A-type and one B-type terminal; in rabbit, these three combinations are found in equal numbers. The current

Figure 2 Cone contacts of an A-type horizontal cell in rabbit.

(a) Drawing of the Lucifer yellow-injected cell. (b) Cone mosaic overlying the cell, revealed by labeling all cones with the marker peanut agglutinin and identifying the S-cones by an S-opsin antiserum. S-cones are shown as triangles and L-cones as circles. Cones contacted by the A-type cell (dendritic field outline given by broken line) are shown as filled symbols and noncontacted cones as open symbols. The cell contacts most of both spectral cone types in its reach. Reproduced from Figure 4 in Hack, I. and Peichl, L. (1999). Horizontal cells of the rabbit retina are non-selectively connected to the cones. European Journal of Neuroscience 11: 2261–2274. With kind permission of Wiley-Blackwell.

view is that mammalian horizontal cells do not participate in color opponency (i.e., the antagonistic interpretation of color opponent channels).

Contacts with rods

Mammalian rods have horizontal cell contacts only with B-type axonal terminals; conversely, B-type axonal terminals connect only to rods. The only reported exception is the cone-dominated gray squirrel retina, where the axon of an H1 cell (B-type equivalent) was described to contact cones and, perhaps, rods. Unlike some other vertebrates, mammals have no type of horizontal cell that is exclusively connected to rods. This role is taken over by the B-type axon terminal system. The B-type axon is not an axon in the functional sense; it does not conduct information from the dendrites and soma to the axon terminal system. The latter is electrically uncoupled from the dendritic part; apparently, the axon is too thin and long (several hundred microns) to conduct the graded potentials that horizontal cells use for signaling. The axonal synaptic connections with the rods resemble those of the dendrites with the cones (Figures 1(e)–1(g)). It is thought that the B-type axon terminal system is the independent rod horizontal cell of mammals. Thus, while being one metabolic entity, the B-type cell represents two functional units. Actually, a small rod input is found in soma recordings of both B- and A-type cells; however, this is attributed to direct rod/cone coupling and not to signal transfer through the B-type axon.

Presumed general pattern in dichromats

S L L S L S L L

A B

ax

Primates (trichromats and dichromats)

Figure 3 Scheme of the cone and rod contacts of horizontal cells in different mammals. (Top) The presumed general connectivity pattern in dichromats, confirmed in rabbit and tree shrew. A- and B-type cells indiscriminately contact S-cones and L-cones; the B-type axon terminal system contacts rods. (Middle) The connectivity pattern in primates. H1 cells contact M- and L-cones, but largely avoid S-cones (broken contact line); their axon contacts the rods. H2 cells contact S-cones strongly (thick contact line) and M/L-cones less strongly; their

axon contacts S-cones, but may also contact M/L-cones.

In dichromatic primates, M- and L-cones are only one spectral L type. (Bottom) The connectivity pattern in horse (and probably other equids). The A-type contacts S-cones exclusively, whereas the B-type makes indiscriminate contacts with S-cones and L-cones. The coloring of horizontal cell somata indicates their spectral tuning. Adapted from Figure 9 in Peichl, L., Sandmann, D., and Boycott, B. B. (1998). Comparative anatomy and function of mammalian horizontal cells. In: Chalupa, L. M. and Finlay, B. L. (eds.) Development and Organization of the Retina: From Molecules to Function, pp. 147–172. New York: Plenum Press. With kind permission of Springer Science and Business Media.

Why are the rods contacted by only one type of process (the B-type axonal terminals) and the cones by two types (A- and B-type dendrites) and what detailed functional difference does that make? An answer is currently not available. The receptive field surround of the ganglion cells becomes broader and shallower with decreasing light levels, and this is interpreted as a useful image-processing strategy. Nevertheless, it is unknown whether and how the rod/horizontal cell connections may be involved.