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

RA Vitreous

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Figure 1 Light micrograph of the retina, choroid, and sclera. The retinal layers are identified as: (a) nerve fiber layer;

(b) ganglion cell layer; (c) inner plexiform layer; (d) inner nuclear layer; (e) outer plexiform layer; (f) outer nuclear layer; (g) rod and cone layer; and (h) retinal pigment epithelial layer. RA, retinal arteriole.

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Figure 2 View from under surface of the human eyeball and optic nerve (ON) showing central artery of the retina (CAR) and its site of penetration into the optic nerve sheath (PPS), medial (MPCA) and lateral (LPCA) posterior ciliary arteries, and ophthalmic artery (OA). Reproduced with permission from Singh (Hayreh), S. and Dass, R. (1960). The central artery of the

retina I. Origin and course. British Journal of Ophthalmology 44: 193–212.

between the retinal arterial and venous pressures. The CRA and cilioretinal artery belong to two arterial systems with different physiological properties. This raises an important physiological issue in eyes with a cilioretinal artery when that eye develops central retinal vein occlusion in which the retinal venous pressure rises suddenly to a high level. The CRA arises directly from the ophthalmic artery and the retinal vascular bed supplied by it has an efficient blood flow autoregulation (see below), so that when there is a fall in perfusion pressure in the retinal arterial bed, caused by a rise in the retinal venous pressure, the autoregulatory mechanism in the central retinal arterial vascular bed kicks in trying to maintain retinal circulation. By contrast, the cilioretinal artery belongs to the choroidal vascular system, which has no autoregulation, so that when the venous pressure rises, there is no corresponding compensatory autoregulatory mechanism. Moreover, the perfusion pressure in the choroidal vascular bed normally is lower than that in the CRA, and there is no corresponding rise of pressure in the choroidal venous bed. In view of all these factors, the following scenario occurs in an eye with cilioretinal artery developing central retinal vein occlusion: sudden occlusion of the central retinal vein results in a

marked rise of intraluminal pressure in the entire retinal capillary bed; when that intraluminal pressure rises above the pressure in the cilioretinal artery, the result is a hemodynamic block in the cilioretinal artery, producing cilioretinal artery occlusion (Figure 7).

Intraretinal Branches of the CRA

Each of the two main branches (superior and inferior) of the CRA at the optic disk usually divides into temporal and nasal branches, which supply the four quadrants of the retina (Figure 4); however, there is marked variation in their vascular pattern. In the retina, the arrangement of the branches and their subdivisions is highly variable, so much so that each eye has a different pattern (Figures 4 and 7). It has been suggested that the pattern could be used for personal identification like a finger print. Usually, there is a dichotomous or right-angle branching pattern. The various branches, by multiple divisions, finally end in terminal or precapillary arterioles, which are usually not visible on ophthalmoscopy. Terminal arterioles play an important role in the regulation of retinal blood flow by constriction or dilatation.

There has been a controversy about the true nature of the arteries in the retina. According to some accounts these are small arteries. Others, however, consider them as arterioles after the first branching in the retina because they possess the following anatomic properties, typically seen in arterioles: (1) the widest part of the lumen of the retinal arterioles is near the optic disk and there its diameter is about 100 mm, which is typically the diameter of an arteriole; and (2) unlike arteries, they possess neither an internal elastic lamina nor a continuous muscular coat. This differentiation from the arteries is important in understanding their pathological involvement in some diseases, such as giant cell arteritis. In the retina, there are no interarterial or arteriovenous anastomoses, so that the retinal vascular bed is an end-arterial system.

These intraretinal arterial branches mainly lie in the nerve fiber and ganglion cell layer, usually under the internal limiting membrane (Figure 1); however, at the arteriovenous crossing they may extend down to the inner nuclear layer.

Retinal Capillary Bed

Each terminal arteriole gives out a plexus of 10–20 interconnected capillaries (Figure 8). Capillaries lie between the feeding arterioles and venules (Figure 8). Around the retinal arteries, there is a capillary-free zone (Figure 8). The retinal capillaries are arranged in two layers (Figure 9): (1) a superficial layer in the ganglion cell and nerve fiber layers, and (2) a deeper layer in the inner nuclear layer which is denser and more complex than the superficial layer. However, in the posterior retina, there may be three layers in the peripapillary region and there is only one layer in the perifoveal region. Furthermore, in the peripheral retina the deep layer disappears and only the superficial layer is left, with a wider network. At the extreme periphery of the retina, there is an avascular zone about 1.5 mm width.

Figure 4 Normal human fundus – left eye.

Figure 5 Ophthalmoscopic appearance of right eye with central retinal artery occlusion (pale retina) and a normal cilioretinal artery in the area of normal retina (arrow).

Figure 6 Fluorescein fundus angiograms of (a) right and (b) left eyes of a person. (a) The right eye has one large cilioretinal artery (one asterisk) supplying the superior one-third of the retina that starts to fill before the central retinal artery (two asterisks) which supplies the rest of the retina. (b) The left eye has two large branching cilioretinal arteries (white) – the one above branches in the upper half and the other below branches in the lower half. This eye lacks a central retinal artery.

Figure 7 Fundus photograph of right eye with nonischemic central retinal vein occlusion associated with cilioretinal artery occlusion (arrow).

Figure 8 Fluorescein fundus angiogram showing retinal vessels and capillary network. A, retinal arteriole; V, retinal vein.

In addition to the retinal capillary bed described above, there is a distinct retinal capillary bed called the radial peripapillary capillaries, which were first described in 1940 (Figures 9 and 10). They have the following special characteristics compared to other retinal capillaries:

1.They are long, straight capillaries, measuring several hundred microns to several millimeters.

2.They form the most superficial layer (Figure 9) lying among the superficial nerve fibers, along the superior and inferior temporal arcades of retinal vessels and the peripapillary region (Figure 10).

3.They rarely anastomose with one another.

4.They arise from the peripapillary retinal arterioles lying deeper in the retina, and drain into retinal venules or veins on the optic disk (Figure 3).

Because of these characteristics, the radial peripapillary capillaries assume importance in the development of several lesions. For example, cotton wool spots are often located in the distribution of the radial peripapillary capillaries, which indicates that the latter may play a role in the pathogenesis of cotton wool spots. In addition, in chronic optic disk edema these capillaries become dilated and develop microaneurysms and hemorrhages.

Figure 9 Schematic representation of two layers of the retinal capillaries and radial peripapillary capillaries (RPC). Reproduced with permission from Henkind, P. (1969). Microcirculation of peripapillary retina. Transactions of the American Academy of Ophthalmology and Otolaryngology 73: 890–897.

Figure 10 Schematic representation of radial peripapillary capillaries. Site of foveola (X). Reproduced with permission from Henkind, P. (1967). Radial peripapillary capillaries of the retina: I. Anatomy: Human and comparative. British Journal of Ophthalmology 51: 115–123.

In the macular region, the capillaries are supplied by arterioles arising from the superior and inferior temporal arteries (Figure 4). Their thickness decreases toward the center of the macula where they are arranged in a single layer. The capillaries are absent in the foveal region, with a capillary-free zone of about 400–500 mm in diameter (Figure 11).

The wall of the retinal capillaries consists of endothelial cells, pericytes, and basement membrane. Their diameter varies from 3.5 to 6 mm. The endothelial cells have

Figure 11 A cast of retinal capillaries in the macular region of a monkey showing the foveal avascular zone in the center. Courtesy of Professor Koichi Shimizu.

tight-cell junctions, which constitute a blood–retinal barrier (see below). In addition to the endothelial cells, there are also pericytes which form a discontinuous layer within the basement membrane of the capillaries. They have a contractile property, by virtue of which they may play a role in regulating blood flow in the capillaries and autoregulation of blood flow (see below). Pericytes are lost preferentially in diabetes so that they may have a role in diabetic retinopathy; it is suggested that diabetic pericyte loss is the result of their migration. Migration of pericytes is also involved in the regulation of angiogenesis.

Retinal Venous Drainage

The postcapillary venules drain the blood from the capillaries but, occasionally, capillaries may join a major vein directly. The terminal arterioles and postcapillary venules are situated in an alternating pattern, with the capillary bed in between the two (Figure 8). The postcapillary venules drain into bigger venules and finally into the branch retinal veins. The lumen of the major branch retinal veins, just before they join to form the central retinal vein, is about 200 mm. In the central part of the retina, the branch retinal veins and arteries usually run in close association and at places cross one another (Figures 4, 5, and 7). On the other hand, in the peripheral retina, the veins do not follow the course of the arteries. Various retinal arteries and veins in the retina cross each other at arteriovenous crossings (Figures 4, 5, and 7). In a study of 189 normal eyes, at the sites of arteriovenous crossing, the artery crossed over the vein in 68% and was the reverse

in the remainder. However, in eyes with branch retinal vein occlusion, the artery crossed over the vein at the site of occlusion in 98% of the cases, indicating that pattern of arteriovenous crossing plays a role in the development of branch retinal vein occlusion. At the site of arteriovenous crossing, the artery and vein share a common fibrous coat and are separated by only a thin endothelial lining and basement membrane.

The superior branch veins usually join to form a superior trunk and the inferior branch veins, an inferior trunk. These superior and inferior trunks join on the optic disk to form the central retinal vein (Figure 3). However, in 20% of eyes, the superior and inferior trunks do not join together at the disk but enter the optic disk as two separate trunks; this represents a congenital anomaly (Figure 12). During the third month of intrauterine life, there are always two trunks of the central retinal vein in the ON, one on either side of the CRA (Figure 13), and one of the two trunks usually disappears before birth; however, in 20% of eyes, a dual-trunked central retinal vein persists into adult life. In such eyes, only one of the two trunks may develop occlusion in the ON, resulting in development of the clinical entity called hemi-central retinal vein occlusion (Figure 12).

The central retinal vein travels in the ON temporal to the artery, where the central retinal vein and artery lie in the center of the ON, surrounded by a fibrous tissue envelope (Figure 14). During its intraneural course, the vein receives many tributaries (Figure 3). The central retinal vein exits the ON and its sheath (Figure 15), and finally drains into either the superior ophthalmic vein or directly into the cavernous sinus.

Nerve Supply

The intraorbital and intraneural portions of the CRA have an adrenergic nerve supply from a sympathetic nerve called the nerve of Tiedemann (Figure 16); however, the retinal branches of the CRA have no adrenergic nerve supply. Therefore, there is no autonomic innervation of the retinal vascular bed.

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Figure 13 Schematic representation of two trunks of the central retinal vein in the anterior part of the optic nerve. A, Arachnoid; C, choroid; CRA, central retinal artery; Col. Br., collateral branches; CRV, central retinal vein; D, dura; LC, lamina cribrosa; OD, optic disc; ON, optic nerve; PCA, posterior ciliary artery; PR, prelaminar region; R, retina; S, sclera; SAS, subarachnoid space.

Figure 12 Fundus photograph of an eye with inferior hemicentral retinal vein occlusion, involving the lower trunk of the central retinal vein. Two trunks (arrows) of the central retinal vein enter the optic disk separately above and below.

Figure 14 Histological sections (Masson’s trichrome staining) showing the central retinal vessels and surrounding fibrous tissue envelope, as seen in a transverse section of the central part of the retrolaminar region of the optic nerve, in a normal rhesus monkey (above) and in a rhesus monkey with experimental arterial hypertension, atherosclerosis and glaucoma (below). CRA ¼Central retinal artery, CRV ¼ central retinal vein;

FTE ¼ fibrous tissue envelope.

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Figure 15 View from under surface of optic nerve in a rhesus monkey showing the intraorbital part of the central retinal vessels and their site of penetration into the sheath of the optic nerve.

CRA, central retinal artery; CRV, central retinal vein; ON, optic nerve; SOV, superior ophthalmic vein. Reproduced with permission from Hayreh, S. S. (1965). Occlusion of the central retinal vessels. British Journal of Ophthalmology 49: 626–645.

Figure 16 Light micrograph of a longitudinal section of optic nerve of a rhesus monkey, showing central retinal artery (CRA) in the center of the nerve and nerve of Tiedemann (arrow) running parallel with the wall of the central retinal artery. (Gros-Schultze’s stain). Reproduced with permission from Hayreh, S. S., Vrabec, Fr. (1966). The structure of the head of the optic nerve in rhesus monkey. American Journal of Ophthalmology 62: 136–150.

Blood–Retinal Barrier

The retina has two types of blood–retinal barriers.

Inner blood–retinal barrier. This lies in the retinal vessels. It is produced by the tight cell junctions between the endothelial cells of the vessels (due to the presence of extensive zonulae occludentes). The tight interendothelial cell junctions block movement of macromolecules from the lumen toward the interstitial space. Pericytes, Mu¨ller cells, and astrocytes also contribute to the proper functioning of this barrier.

Outer blood–retinal barrier. Tight cell junctions between the retinal pigment epithelial cells (Figure 1) also produce a blood–retinal barrier, preventing the leakage of fluid from the choroid into the retina. This barrier breaks down when the retinal pigment epithelial cells are destroyed or subjected to ischemia, as in hypertensive choroidopathy.

The blood–retinal barrier plays an important role in the regulation of the microenvironment in the retina. An intact blood–retinal barrier is essential for maintaining retinal structure and function. Breakdown of this barrier results in increased vascular permeability of the capillaries, which causes retinal edema, as seen in a variety of retinopathies. Breakdown of the inner blood–retinal barrier may be caused by acute distension of the vessel walls, ischemia, chemical influences, defects in the endothelial cells, or failure of the active transport system.

The retinal tissue itself has no barrier in its stroma, therefore fluid may diffuse from one part to the adjacent areas.

Autoregulation of Retinal Blood Flow

The object of blood flow autoregulation in a tissue is to maintain relatively constant blood flow during changes in perfusion pressure. This is an important mechanism to regulate blood flow. The retinal circulation has efficient autoregulation. The exact mechanism and site of autoregulation are still unclear except that it most probably operates by altering the vascular resistance. It is generally considered as a feature of the terminal arterioles; so with the rise or fall of perfusion pressure beyond normal levels, the terminal arterioles constrict or dilate, respectively, to regulate the vascular resistance and thereby the blood flow. Recent studies have suggested that pericytes in the retinal capillaries play a role in autoregulation as well because of their contractile property. The metabolic needs of the tissue also regulate the autoregulation. Autoregulation works within a critical range of perfusion pressure, and it breaks down with any rise or fall of the perfusion pressure beyond the critical autoregulatory range.

The vascular endothelium plays an active role in the vasomotor function of both macroand microvasculatures,

including maintenance of vascular tone and regulation of blood flow. Recent studies suggest that vascular-endothe- lial-derived vasoactive agents (e.g., endothelin-1, thromboxane A2, and prostaglandin H2 – vasoconstrictors; and nitric oxide – a vasodilator) profoundly modulate local vascular tone and, thereby, may also play a role in autoregulation. Mechanical stretching and increases in arteriolar transmural pressure induce the endothelial cells to release contracting factors affecting the tone of arteriolar smooth muscle cells and pericytes. Therefore, damage to vascular endothelium (as in arteriosclerosis, atherosclerosis, hypercholesterolemia, aging, diabetes mellitus, ischemia, and possibly from other causes) may be associated with abnormalities in the production of endothelial vasoactive agents, and consequent autoregulation abnormalities.

See also: Blood–Retinal Barrier; Breakdown of the Blood–Retinal Barrier; Breakdown of the RPE Blood– Retinal Barrier; Central Retinal Vein Occlusion; Pathological Retinal Angiogenesis.

Further Reading

Anderson, D. R. (1996). Glaucoma, capillaries and pericytes 1. Blood flow regulation. Ophthalmologica 210: 257–262.

Cunha-Vaz, J. G. (1976). The blood–retinal barriers. Documenta Ophthalmologica 41: 287–327.

Duke-Elder, S. and Wybar, K. C. (1961). Anatomy of the visual system. In: Duke-Elder, S. (ed.) System of Ophthalmology vol. 2, pp. 363–382. London: Kimpton.

Haefliger, I. O., Meyer, P., Flammer, J., and Lu¨scher, T. F. (1994). The vascular endothelium as a regulator of the ocular circulation: A new concept in ophthalmology? Survey of Ophthalmology

39: 123–132.

Hayreh, S. S. (1963). The cilio-retinal arteries. British Journal of Ophthalmology 47: 71–89.

Hayreh, S. S. and Hayreh, M. S. (1980). Hemi-central retinal vein occlusion. Pathogenesis, clinical features, and natural history.

Archives of Ophthalmology 98: 1600–1609.

Hayreh, S. S., Fraterrigo, L., and Jonas, J. (2008). Central retinal vein occlusion associated with cilioretinal artery occlusion. Retina 28: 581–594.

Henkind, P. (1967). Radial peripapillary capillaries of the retina: I. Anatomy: Human and comparative. British Journal of Ophthalmology 51: 115–123.

Henkind, P. (1969). Microcirculation of peripapillary retina. Transactions of the American Academy of Ophthalmology and Otolaryngology

73: 890–897.

Justice, J. Jr. and Lehmann, R. P. (1976). Cilioretinal arteries. A study based on review of stereo fundus photographs and fluorescein angiographic findings. Archives of Ophthalmology 94: 1355–1358.

Kaur, C., Foulds, W. S., and Ling, E. A. (2008). Blood–retinal barrier in hypoxic ischaemic conditions: Basic concepts, clinical features and management. Progress in Retinal and Eye Research 27(6):

622–647.

Pournaras, C. J., Rungger-Bra¨ndle, E., Riva, C. E., Hardarso, S. H., and Stefansson, E. (2008). Regulation of retinal blood flow in health and disease. Progress in Retinal and Eye Research 27: 284–330.

Singh (Hayreh), S. and Dass, R. (1960). The central artery of the retina I. Origin and course. British Journal of Ophthalmology 44: 193–212.

Singh (Hayreh), S. and Dass, R. (1960). The central artery of the retina II. Distribution and anastomoses. British Journal of Ophthalmology 44: 280–299.

Weinberg, D., Dodwell, D. G., and Fern, S. A. (1990). Anatomy of arteriovenous crossings in branch retinal vein occlusion. American Journal of Ophthalmology 109: 298–302.

Wise, G. N., Dollery, C. T., and Henkind, P. (1971). The Retinal Circulation, pp. 20–54. New York: Harper and Row.

Glossary

Ca2þ microdomains – Local submembrane regions of elevated intracellular Ca2þ caused by the influx of Ca2þ through nearby Ca2þ channels.

Calcium-induced calcium release (CICR) –

Release into the cytoplasm of Ca2þ ions stored in the endoplasmic reticulum that is triggered by the Ca2þ-dependent activation of ryanodine receptors.

ERG b-wave – The electroretinogram (ERG) is a massed electrical response of the retina to light that can be recorded by electrodes on the surface of the cornea. ON bipolar cells provide the source of the b-wave of the ERG. A selective reduction in the b-wave indicates a reduction in signaling between photoreceptors and ON bipolar cells.

L-type calcium currents – Currents from high- voltage-activated CaV1.1 (alpha 1S), CaV1.2 (alpha 1C), CaV1.3 (alpha 1D), or CaV1.4 (alpha 1F) calcium channels which show sustained activation and can be selectively blocked by dihydropyridine antagonists.

OFF bipolar cells – Second-order retinal bipolar cells which exhibit a hyperpolarizing response to light mediated by non-NMDA ionotropic glutamate receptors.

ON bipolar cells – Second-order retinal bipolar cell subtypes which exhibit a depolarizing response to light mediated by mGluR6 metabotropic glutamate receptors.

Readily releasable pool – A pool of synaptic vesicles primed for rapid release by elevation of intracellular calcium.

SNARE proteins – SNAP and NSF attachment receptors are a family of proteins participating in vesicle fusion. They can be subdivided into vesicle SNAREs (v-SNAREs), which attach to vesicles, and target SNAREs (t-SNAREs), which associate with the plasma membrane.

Synaptic ribbon – An electron dense presynaptic structure that tethers synaptic vesicles in nerve terminals of sensory neurons including photoreceptors, retinal bipolar cells, hair cells, pinealocytes, and electroreceptors.

Total internal reflectance (TIRF) microscopy – By taking advantage of the subwavelength evanescent

field of light created by reflections at the interface between a cell and coverslip, TIRF microscopy can be used to visualize subwavelength structures such as synaptic vesicles.

Anatomy of the Ribbon Synapse

Structures of the electron dense ribbons found at the synapses of retinal photoreceptors and bipolar cells differ depending on cell type. In cross section, photoreceptor ribbons appear as 35 nm thick bars, but in three dimensions they form flat, ribbon-like structures. Mammalian rods have one to two ribbons that can be up to 2 mm in length and extend up to 1 mm into the cytoplasm. Each wraps around the synaptic ridge to form crescent or horseshoe shapes. The ribbons in mammalian cone are smaller, typically less than 1 mm long and extend only a few hundred nanometers into the cytoplasm. They are shaped like surfboards, and typically a dozen or more ribbons are in each cone terminal. Sitting just below the photoreceptor ribbon is a trough-like arciform density. Ribbons in bipolar cells are planar, like those in rods, but they are smaller than both rod and cone ribbons.

The focus of this review is on ribbon synapses in retina; however, ribbon synapses are not unique to retina. They are present in other sensory neurons, including pinealocytes, electroreceptors in the lateral line organ of fishes, and hair cells of the cochlea and vestibular apparatus. Again, the ribbons in the synapses of these cells have different structures depending on cell type. For example, hair cell ribbons are small spheres.

The ribbon synapses of photoreceptor terminals contain many more synaptic vesicles than conventional synapses. Conventional synapses have 10–100 vesicles near each presynaptic density compared to the synaptic terminal of a lizard cone, which contains 170 000 vesicles or 7000 vesicles per ribbon. Furthermore, 85% of the vesicles at ribbon synapses are freely mobile and readily participate in release compared to 20% of vesicles at conventional synapses. The expanded mobility of vesicles at ribbon synapses may be due to the absence of synapsins which have been proposed to tether synaptic vesicles at conventional synapses. The small subset of vesicles in photoreceptor terminals that are tethered to synaptic ribbons are attached by fine filaments (Figure 1).

Vesicle Pools and Vesicular Release at

Synaptic Ribbons

Newly tethered vesicles can be found throughout the ribbon, indicating that vesicles either freely enter the ribbon at any position or redistribute rapidly about the ribbon face after attachment. However, once attached to the ribbon, vesicles exit only from the base. Vesicles tethered on the bottom first to third rows of the ribbon contact the plasma membrane along the synaptic ridge. These vesicles constitute a pool that can be released rapidly in response to increased Ca2þ levels. In bipolar cells, the fastest component of vesicular release equals the number of vesicles tethered along the bottom row of the ribbon, and maintained depolarization stimulates release of the total number of vesicles lining the entire ribbon. Similarly, the total releasable pool in rod photoreceptors equals the number of vesicles tethered to the entire ribbon ( 700 vesicles in amphibian rods), and there is an ultrafast component similar to the number of vesicles tethered at the ribbon base ( 30 vesicles). Because of their smaller size, cone ribbons have a smaller releasable pool than rod ribbons.

In contrast with bipolar and photoreceptor cells, the ultrafast release component in hair cells exceeds the number of vesicles lining the bottom row by nearly 10-fold, and the total releasable pool exceeds the number of vesicles lining the ribbon sixto eightfold. The large ultrafast or rapidly releasable pool could be explained by compound fusion between adjacent vesicles as discussed later. The large size of the total pool suggests that vesicles released from the ribbon are replenished rapidly from the surrounding cytoplasm.

Vesicular transmitter release clearly occurs at the ribbons in ribbon synapses, but the ribbons may not be the only site of transmitter release. Photoreceptors also

contact bipolar cell dendrites at flat or basal junctions identified by preand postsynaptic membrane densities. In mammalian retina, the basal junctions of cones only contact OFF-type bipolar cells and the dendrites of many OFF-type bipolar cells are not directly apposed to synaptic ribbons. In salamander retinas, the architecture of photoreceptor input to bipolar cells is different, and OFF bipolar cells receive 80% of their contacts from ribbon synapses and only 20% from basal junctions. On the other hand, salamander ON-type bipolar cells receive20% of their contacts from ribbons synapses and 80% from basal junctions.

Basal junctions lack the vesicle clusters which typify conventional synapses; however, the absence in mammals of direct ribbon contacts onto many OFF bipolar cells led to the suggestion that they necessarily receive synaptic input from basal junctions. This idea was tested in experiments that compared synaptic events recorded simultaneously from two OFF bipolar cells – one that contacted photoreceptors only at basal junctions and a neighbor whose dendrites approach the ribbon. These studies showed that glutamate released at a synaptic ribbon can diffuse rapidly to bipolar cell processes at basal junctions. This finding shows that glutamate released at the ribbon can reach basal junctions, although it does not exclude the possibility of additional nonribbon release events.

The question of whether nonribbon release events occur in ribbon synapses has been addressed further by using total internal reflectance (TIRF) microscopy to visualize single-vesicle fusion events. Studies of release from bipolar cells indicate that up to 1/3 of fusion events occur at ectopic sites away from the ribbon including much of the release during sustained depolarization. Studies on hair cells from mice with disrupted ribbon anchoring also suggest a role for nonribbon sites in

sustained release by showing that sustained release is unchanged although fast release is diminished. However, in photoreceptors, as discussed later, there is evidence suggesting that sustained release occurs predominately at the ribbon.

Role of the Ribbon in Release

The functions of the ribbon in release are not fully understood. It has been widely suggested that the ribbon may operate like a conveyor belt, acting as a molecular motor to accelerate delivery of vesicles to their release sites. Consistent with this possibility is the presence of a kinesin motor protein, KIF3A, at the ribbon. However, release of vesicles attached to the ribbons does not require ATP, although ATP is needed for subsequent replenishment and priming of vesicles. Ribbons are also not necessary for sustaining high-frequency release since it can be observed at conventional central nervous system (CNS) synapses without ribbons. In fact, during sustained release from photoreceptor ribbons, the delivery of vesicles to the base of the ribbon is actually slower than the rate predicted from delivery by simple diffusion. Thus, rather than accelerating release, synaptic ribbons in cones appear to slow the rates of sustained release by constraining the rate of vesicle delivery to the base. By making release more regular, such a mechanism could improve the ability to detect small intensity changes that produce small changes in release rate.

Another hypothesis of ribbon function is that it may operate as a vesicle trap. In this scenario, vesicles moving about the terminal by Brownian motion occasionally collide with the ribbon face where they are captured like flies to fly paper and then delivered to the base for release.

Ribbons may assist in vesicle priming. The match between releasable pool size and the number of vesicles tethered to rod or bipolar cell ribbons indicates that tethered vesicles are primed for release. Furthermore, members of the RIM family of proteins involved in vesicle priming localize to the ribbon. RIM proteins are synaptic proteins required for normal neurotransmitter release.

Another proposed role for the ribbon is to facilitate compound fusion during depolarization by promoting fusion between neighboring vesicles on adjacent rows of the ribbon. Compound fusion could explain the finding in hair cells that many more vesicles fuse rapidly after stimulation than are anchored at the base of the ribbon. The possibility of compound fusion in hair cells is supported by statistical properties of fluctuations in postsynaptic currents and presynaptic exocytotic capacitance changes. There is also ultrastructural and electrophysiological evidence for compound or coordinated fusion of multiple vesicles at bipolar cell synapses.

Ribbons may perform more than one of these functions. They may capture vesicles, prime them for release, regulate their delivery to release sites, and facilitate compound fusion. To sort out the unique functions of ribbons and ribbon synapse, the biochemistry and electrophysiological properties of ribbon synapse are being studied in detail.

Synaptic Proteins

Ribbon synapses contain many of the same proteins as conventional synapses. For example, conventional and ribbon synapses both employ SNARE proteins in vesicle fusion: synaptobrevin (VAMP1 and 2), SNAP-25, and syntaxin. Ribbon synapses also possess Munc 18-1, Munc 13-1, RIM, and rab3A proteins which assist with assembly of the SNARE complex and vesicle priming. RIM1 is distributed across the ribbon, whereas RIM2 localizes to the ribbon base, suggesting that they may have different roles.

In addition to many similarities, there are also differences among the proteins at ribbon and conventional synapses. In place of syntaxin-1 found at many conventional synapses, ribbon synapses utilize syntaxin-3b. In place of complexins 1 and 2 that interact with the SNARE complex at many synapses, ribbon synapses possess complexins 3 and 4. Ribbon synapses lack synapsins. The functional consequences of these differences remain to be explained.

As in conventional synapses, a rise in intracellular free Ca2þ is required for neurotransmitter release, but the identity of the calcium sensor(s) at ribbon synapses is unsettled. Synaptotagmin I is the principal calcium sensor at most conventional synapses. Antibodies to synaptotagmin I/II label photoreceptor and bipolar cell ribbon synapses in mouse and bovine retina but do not label these synapses in goldfish and salamander retina, which are instead labeled by antibodies to synaptotagmin III. In hair cells, it has been proposed that otoferlin, not synaptotagmin, is the principal calcium sensor for exocytosis.

The most conspicuous difference between ribbon and conventional synapses is the presence of the ribbonspecific protein, ribeye. Ribbons are constructed from interdomain interactions between adjoining ribeye molecules. Each bipolar cell ribbon is formed from 4000 ribeye molecules and the 10-fold greater surface area of rod ribbons suggests that they are built from 40 000 ribeye molecules. Ribeye is an alternative transcript of the gene for transcriptional repressor C-terminal-binding protein 2 (CtBP-2) with a unique ribbon-specific A domain and an enzymatic B domain. Interactions between ribeye molecules are regulated by NAD and NADH levels, suggesting a mechanism by which changes in the metabolic state of the terminal could contribute to observed circadian changes in ribbon structure.