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

new connective tissue in an effort to increase the mechanical strength of the lamina. Addition of material to the posterior surface of the lamina ensures, at least in the short term, that axons will be minimally damaged in the process. Ultimately, this process appears to be a losing battle, as axons become progressively more damaged and begin to degenerate, the optic nerve head becomes a series of collapsed connective tissue plates. A critical question in the pathophysiology of glaucoma is what ultimately causes the damage to the axons in this region.

Several theories have been postulated for the source of the axonal damage. The earliest of these were contradictory models that postulated either (1) a decrease in blood flow

to the optic nerve head, creating an ischemic environment for the ganglion cell axons, or (2) direct mechanical damage to the axons as they passed through the connective tissue plates of the lamina such that they were being crimped as the plates underwent the process of posterior deformation. More recent models have predicted that the altered function of the astrocytes and microglia in this region leads to the release of toxic molecules. Several studies have suggested that these toxins could range from highly reactive nitric oxide radicals mediated by the activity of nitric oxide synthase-(NOS)-2 to tumor necrosis factor a (TNFa), which is produced by astrocytes under stress conditions and can activate retinal ganglion

cell death. Regarding this hypothesis, although many questions remain unanswered, experimental evidence is clear that astrocytes, microglia, and possibly LC cells become reactive in the glaucomatous optic nerve head (Figure 3), leading to molecular and behavioral changes that have been associated with pathology in several other neurodegenerative disorders.

Others, however, have reasoned that the initial responses of glia in the nerve head are just as likely trying to protect the axons as they compensate for the increased mechanical strain. Protective effects of astrocytes can include becoming a source of trophic factors for damaged neurons. In addition, they also act as the principal source of glycogen stores in the central nervous system (CNS), and are typically involved in monitoring and balancing extracellular pH and ionic conditions, which both support neurons and prevent damaging effects. Thus, central questions in our understanding of glaucoma pathology

are whether the optic nerve glia simply become unable to continue their role in supporting the ganglion cell axons, or do they instead change to a molecular behavior that contributes to axonal pathology?

Another important consideration in our understanding of the effects of elevated IOP on the optic nerve head is the differential between IOP and arteriolar blood pressure. From a biomechanical standpoint, perfusion pressure to the small capillaries in the nerve head could meet much higher resistance in the face of elevated IOP. Thus, it is reasonable to predict that reduced blood flow would lead to micro-ischemic environments in this region. A similar consideration could also be made for the process of axoplasmic transport, particularly retrograde transport. Taken together, the pressure differential encountered at the lamina of a glaucomatous eye may consummate in a catastrophic stress where neurons are consuming greater amounts of adenosine triphosphate (ATP) to maintain

axoplasmic transport in the face of reduced access to nutrients and oxygen from both compromised astrocytes and reduced arteriolar blood flow. Ultimately, a scenario may develop where energy supplies are no longer sufficient to sustain transport against the pressure gradient and the axon is forced to execute a self-destruct pathway.

Compartmentalized Self-Destruct

Pathways and the Pathology of Glaucoma

A major factor in understanding how elevated IOP leads to ganglion cell death is understanding how ganglion cells die in the first place. Ganglion cells are projection neurons of the CNS. The cell body, or soma, is located in the inner retina, where they take input from neurons in the outer retina through a complex network of dendrites located in the inner plexiform layer. The soma then projects an axon through the nerve fiber layer, which is adjacent to the vitreous, exiting the eye through the lamina into the optic nerve. In simple terms, a ganglion cell has four distinct compartments: the soma, the axon, a synapse at the end of the axon, and the dendritic tree.

Recently, the death of retinal ganglion cells has been evaluated in the context of effects on their individual compartments. Several studies, using genetically engineered mice, in which mutations in specific genes selectively affect the degenerative process of individual compartments, have shown that ganglion cell death can occur in an autonomous, compartment-specific, fashion. Two mutations, in particular, have contributed to the understanding of the process of ganglion cell death in glaucoma. The first of these is a knock-out mutation created in a gene called Bax, which is a member of a larger gene family that regulates the intrinsic apoptotic pathway by affecting permeability changes and dysfunction in mitochondria. Although apoptosis is generally considered in the same context as cell death, the term actually describes the self-destruct pathway executed by cell somas. In a defined, naturally occurring form of glaucoma in the DBA/2J inbred line of mouse, Bax deficiency was found to completely abrogate ganglion cell soma loss, but not the degeneration of the ganglion cell axons. A second mutant gene, which occurs naturally in mice, is called Wallerian degeneration slow (Wld S). This mutant gene dramatically reduces the degenerative process called Wallerian degeneration, which is the self-destruct pathway executed by dying axons. In DBA/2J mice carrying the WldS mutation, ganglion cell axons are dramatically preserved in the face of ocular hypertension that causes glaucoma in wild-type mice. Importantly, in these mice, ganglion cell somas are also spared compared to wild-type animals. These data provide insight on the timing of events associated with ganglion cell loss. They indicate that axon loss is not prevented by blocking soma loss,

while soma loss can be attenuated if axon loss is prevented. Together, this strongly implicates glaucoma as an axogenic disease, distinct from other neurodegenerative disorders where the soma is the fist site of injury. Supporting this model, all studies examining the timing and progression of both optic nerve degeneration and retinal disease in the DBA/2J mouse (Figure 4), point to axonal damage preceding the loss of ganglion cell somas.

In a scheme that links elevated IOP to ganglion cell damage and death, the activation of an autonomous selfdestruct pathway in the ganglion cell axon will, in turn, lead to the activation of the autonomous apoptotic pathway in the ganglion cell soma. The mechanism by which this occurs is also unknown, but several studies suggest that the loss of axonal transport will reduce the flow of neurotrophic growth factors to ganglion cell somas. This model for the activation of soma death in glaucoma has been coined the neurotrophin hypothesis. During development, the normal pruning of retinal ganglion cells, in which approximately 50% of the cells are eliminated, coincides with an increased dependence on neurotrophic factors for survival. Similarly, application of exogenous neurotrophins, after acute or chronic damage to the optic nerve, is able to prolong ganglion cell survival.

Although these studies are consistent with the neurotrophin hypothesis, there are several caveats that require further explanation and reveal the need for continued study. First, the level of protective effect of exogenous neurotrophins is transient, even if efforts are made to introduce a continuous supply of any given factor over a long period. Second, injury to the optic nerve is known to stimulate the expression of endogenous trophic factors by other cells, particularly macroglia, in the retina, leading to the question of why this increase is not an expression sufficient to sustain ganglion cell somas in the absence of retrograde transport? Part of the reason for this effect may lie within molecular changes occurring in the ganglion cell somas once they have become injured. An early component of the injury process is the silencing of normal gene expression and some of the genes that are downregulated appear to be for the receptors that neurotrophic factors bind to. An alternative explanation is that many of the studies showing the transient effects of exongenous trophic factors are usually examining the effects of only a single factor at a time. Compelling available data suggest that treatments that elicit the infiltration of cells, such as macrophages, provide a much greater and longer-term effect on the survival of ganglion cell somas. In this case, infiltrating cells are likely producing and secreting much more complex mixtures of trophic factors than could be applied by experimental manipulation. Lastly, the signaling pathways activated by neurotrophic factors are variable and highly regulated. One such level of regulation is by the localization of target receptors on neurons, such that receptors bound at the synapse are likely to activate distinct mitogen-activated protein kinase

(MAPK) pathways than the same receptors if they interact with a neurotrophin ligand at the plasma membrane of the cell soma. The activation of distinct MAPK pathways could certainly yield different effects on ganglion cell survival. This was demonstrated by recent studies showing increased survival and gain of function of ganglion cells in cat eyes after a partial optic nerve crush, when the neurotrophic factor, brain-derived neurotrophic factor (BDNF), was applied both as an intravitreal injection and simultaneously infused into the visual cortex.

Acknowledgments

The author would like to thank Dr. Harry A. Quigley for contributing scanning electron microscope images of the human lamina cribrosa and Dr. Cassandra L. Schlamp for

preparing the figures. Some of this work was funded by NIH grant R01EY12223 to the author.

See also: Animal Models of Glaucoma; Retinal Ganglion Cell Apoptosis and Neuroprotection.

Further Reading

Hernandez, M. R. (2000). The optic nerve head in glaucoma: Role of astrocytes in tissue remodeling. Progress in Retinal and Eye Research 19: 297–321.

Hernandez, M. R., Andrzejewska, W., and Neufeld, A. (1990). Changes in the extracellular matrix of the human optic nerve head in primary open-angle glaucoma. American Journal of Ophthalmology 102:

180–188.

Howell, G. R., Libby, R. T., Jakobs, T. C., et al. (2007). Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma. Journal of Cell Biology 179: 1523–1537.

Johnson, E. C., Guo, Y., Cepurna, W. O., and Morrison, J. C. (2009). Neurotrophin roles in retinal ganglion cell survival: Lessons from rat glaucoma models. Experimental Eye Research 88: 808–815.

Johnson, E. C., Morrison, J. C., Farrell, S., et al. (1996). The effect of chronically elevated intraocular pressure on the rat optic nerve head extracellular matrix. Experimental Eye Research 62: 663–674.

Libby, R. T., Li, Y., Savinova, O. V., et al. (2005). Susceptibility to neurodegeneration in glaucoma is modified by Bax gene dosage.

PLoS Genetics 1: 17–26.

Nickells, R. W., Semaan, S. J., and Schlamp, C. L. (2008). Involvement of the Bcl2 gene family in the signaling and control of retinal ganglion cell death. Progress in Brain Research 173: 423–435.

Quigley, H. A., Addicks, E. M., Green, W. R., and Maumenee, A. E. (1981). Optic nerve damage in human glaucoma: II. The site of injury and susceptibility to damage. Archives of Ophthalmology 99: 635–649.

Roberts, M. D., Grau, V., Grimm, J., et al. (2009). Remodeling of the connective tissue microarchitecture of the lamina cribrosa in early experimental glaucoma. Investigative Ophthalmology and Visual Science 50: 681–690.

Schlamp, C. L., Li, Y., Dietz, J. A., Janssen, K. T., and Nickells, R. W. (2006). Progressive ganglion cell loss and optic nerve degeneration

in DBA/2J mice is variable and asymmetric. BMC Neuroscience 7: 66.

Schwartz, M., Yoles, E., and Levin, L. A. (1999). ‘Axogenic’ and ‘somagenic’ neurodegenerative diseases: Definitions and therapeutic implications. Molecular Medicine Today 5: 470–473.

Soto, I., Oglesby, E., Buckingham, B. P., et al. (2008). Retinal ganglion cells downregulate gene expression and lose their axons within the optic nerve head in a mouse glaucoma model. Journal of Neuroscience 28: 548–561.

Tezel, G., Hernandez, M. R., and Wax, M. B. (2001). In vitro evaluation of reactive astrocyte migration, a component of tissue remodeling in glaucomatous optic nerve head. Glia 34: 178–189.

Whitmore, A. V., Libby, R. T., and John, S. W. M. (2005). Glaucoma: Thinking in new ways – a role for autonomous axonal self-destruction and compartmentalised processes? Progress in Retinal and Eye Research 24: 639–662.

Yang, Z., Quigley, H. A., Pease, M. E., et al. (2007). Changes in gene expression in experimental glaucoma and optic nerve transection: The equilibrium between protective and detrimental mechanisms. Investigative Ophthalmology and Visual Science 48: 5539–5548.

Introduction

The vertebrate retina is a laminated tissue in which the various component cell types and their synaptic connections are arranged in distinct layers or laminae. This laminar organization, together with a high correspondence between structure and function, makes the vertebrate retina an excellent model system for studying the development of neuronal circuits. The development of retinal circuits generally follows the same sequence of major events for all vertebrates studied thus far. In this article, we provide an overview of the assembly of circuits in the vertebrate retina, primarily focusing on the mouse and zebrafish that have in recent years become key model systems because of the availability of transgenic animals. Circuit assembly involving retinal neurons requires that each cell type forms connections with their appropriate synaptic partners and establishes the correct density of synaptic connections with these synaptic partners. We first briefly review the overall organization of the mature retinal circuitry and then provide the current views on how these circuits are assembled during the development.

Organization of Retinal Circuits of

Vertebrates

Figure 1 shows the general morphological organization of the mature vertebrate retina. Cone photoreceptors transmit light-evoked signals to bipolar cells that relay these signals to the output neurons of the retina, the retinal ganglion cells. There are two major functional subclasses of bipolar cells: ON bipolar cells are depolarized by increased illumination, whereas OFF bipolar cells are hyperpolarized. Cone photoreceptors contact cone bipolar cells whereas in general, rod photoreceptors contact a single class of rod bipolar cells, which form a circuit dedicated to low light vision (the rod pathway). Unlike cone bipolar cells, rod bipolar cells do not contact retinal ganglion cells directly, and instead contact AII amacrine cells. Neurotransmission along the vertical pathway comprising photoreceptors–bipolar cells–retinal ganglion cells is mediated by glutamate. Signals transmitted along the vertical pathway are modulated by amacrine interneurons that provide feedforward inhibition directly onto retinal ganglion cells and feedback inhibition onto bipolar cells. In the inner plexiform layer (IPL), lateral inhibition is mediated by either gamma aminobutyric acid

(GABA)-ergic or glycinergic transmission from the amacrine cells. In the outer plexiform layer (OPL), photoreceptor transmission is modulated by horizontal cells, although the exact mechanism(s) by which this modulation occurs remains highly debated.

Formation of Retinal Synaptic Laminae

The exquisite lamination of cell bodies and synaptic connections of the vertebrate retina have long attracted investigations into the mechanisms that organize this orderly arrangement. Lamination first emerges in the inner retina where retinal ganglion cells, the first-born neurons, migrate to the basal surface of the retina after cell division at the apical surface. As a ganglion cell layer forms, amacrine interneurons are generated and migrate to the inner retina where the majority of these cells accumulate and form a cell layer next to the ganglion cell layer. As amacrine cells and ganglion cells elaborate their processes, an IPL takes shape. An OPL becomes apparent later as the axons and dendrites of the photoreceptors, bipolar cells and horizontal cells differentiate and become confined to a narrow lamina. Interestingly, horizontal cells differentiate early, at around the time of amacrine and ganglion cell genesis, but they do not reach their laminar position until after the IPL emerges. This sequence of lamination, from inner to outer, thus does not depend strictly on the time course of cell generation and the subsequent migration of the various cell types (summarized in Figure 2). The lamination of the axons and dendrites of each cell type of the retina is described in detail in the following sections in the context of circuit assembly.

Assembly of the Vertical Pathway

The vertical pathway of the retina is the major excitatory pathway, conveying light information from the outer to inner retina through glutamatergic transmission. This pathway from photoreceptors to bipolar cells to retinal ganglion cells develops later than circuits involving lateral connections from amacrine cells and horizontal cells.

In order to understand how vertical circuits in the retina are assembled, many studies have focused on the mechanisms that organize the presynaptic axons and postsynaptic dendrites of cellular components of the vertical pathway into their appropriate laminae. How the axonal

R C C R C

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and dendritic arbors of retinal ganglion cells, bipolar cells, and photoreceptors develop has largely been investigated by imaging labeled cells by light microscopy and by reconstructions from serial electron microscopy. Despite the static nature of the observations, these studies have collectively provided insightful views of how retinal ganglion cells, bipolar cells, and photoreceptors acquire the mature forms of their neuronal processes. More recently, live imaging approaches have captured the dynamic changes in axonal and dendritic architecture of developing retinal neurons, including the retinal ganglion cells and bipolar cells. These time-lapse studies confirmed previous observations and also revealed surprising cellular behaviors resulting in the stratified processes of some types of ganglion cells and bipolar cells.

Retinal Ganglion Cells

Dendritic stratification of retinal ganglion cells has been studied extensively in a variety of species. The stratification level of retinal ganglion cell dendrites is correlated with the type of functional input these neurons receive from cone bipolar cells. As indicated in Figure 1, retinal ganglion cells that depolarize to increased illumination have dendritic arbors stratifying in the inner three-fifths of the IPL, whereas ganglion cells stratifying in the outer two-fifths of the IPL hyperpolarize to illumination. In mammalian retinas including cat, rabbit, ferret, mouse, rat, and quokka, immature retinal ganglion cells have dendrites that course through the entire depth of the forming IPL (Figure 3(a)). This initially diffuse arrangement is altered with maturation, giving rise to laminated arbors that stratify at depths unique to each subtype of retinal ganglion cell. Such studies have largely focused on ganglion cells that later possess a single monostratified arbor. Retinal ganglion cells that have two stratified arbors

in mammalian retina (the bistratified ganglion cells) have not been followed throughout their early differentiation, mainly because of a lack of cell-specific markers for ganglion cell subtypes prior to when their dendrites stratify.

In zebrafish, a large proportion of retinal ganglion cells possess more than one stratified dendritic arbor. Because in vivo time-lapse imaging of individual retinal ganglion cells is possible in the transparent zebrafish, the sequence of events leading to the stratification of ganglion cell dendrites can be monitored. Such imaging studies demonstrated that ganglion cells with multiple stratified arbors appear to form each arbor, one stratum at a time (Figure 3(b)). These observations in zebrafish raise the possibility that dendrites in mammals and fish may adopt distinct mechanisms to achieve their stratification patterns. Alternatively, retinal ganglion cells that have multiple stratified arbors may develop differently from cells that become monostratified. Thus, future experiments in which bistratified ganglion cells in mammalian retinas can be identified early in their development are needed to distinguish between these possibilities.

What regulates retinal ganglion cell dendritic stratification? Studies in cat and ferret suggested that blockade of neurotransmission affects the emergence of stratified dendritic arbors of the retinal ganglion cells. Chronic application of the amino-phosphonobutyric acid (APB) agonist of the metabotropic glutamate receptor 6 (mGluR6) receptor found on the dendrites of mature ON bipolar cells led to a failure of ganglion cell dendrites to become stratified. However, APB has been shown to suppress the activity of all retinal ganglion cells in the immature ferret retina and thus its site of action is unclear. Examination of mice in which transmission along the vertical pathway is expected to be perturbed led to different observations. In mice lacking

the mGluR6 receptor, ganglion cell dendritic stratification is normal – in this case, however, one might expect that spontaneous release of neurotransmitter from the bipolar cells is unaffected even though visual stimulation along the ON pathway is abolished. Numerous studies have since been carried out to assess the role of visual experience in regulating dendritic stratification of retinal ganglion cells. Dark-rearing appears to decrease the developmental reduction in the proportion of ON–OFF retinal ganglion cells in mice. This developmental reduction is also less prevalent in mice lacking glycine receptors. In contrast, factors independent of activity have been found to guide dendritic lamination of ganglion cells. In particular, recent studies of the chick retina showed that members of the superfamily of immunoglobulin family of adhesion molecules (sidekicks and DsCam) influence the lamination patterns of retinal ganglion cells and amacrine cells. Although not yet completely worked out, it appears that molecular guidance cues involving adhesion molecules initially guide stratification of ganglion cell dendrites but later visually evoked activity may continue to shape the final lamination patterns of the ganglion cell dendrites.

Bipolar Cells

The development of the axonal terminals of bipolar cells has mostly been studied in a variety of species by single cell labeling methods using Golgi techniques and immunolabeling. Golgi-labeled cells that were presumed to be bipolar cells based on their radial morphology and terminal ending in the IPL appear to confine their axon terminals to specific depths within the IPL even before these cells form dendrites. Immunostaining for recoverin also suggested that ON and OFF bipolar cells stratify early in their differentiation. In transgenic mice, the mGluR6 promoter drives expression of green fluorescent protein in a subset of bipolar cells (ON bipolar cells). Expression is observed when these cells still possess apical and basal processes that are attached to the outer and inner limiting membranes, respectively. Time-lapse imaging of the retinas from these transgenic mice unequivocally demonstrated that axons and dendrites extend from the neuroepithelial-like processes as the bipolar cells differentiate (Figure 4(a)). Whether axons and dendrites in other species including zebrafish also develop in a similar manner is yet to be determined. Both axons and dendrites of the mouse ON bipolar cells explore retinal

depths outside their final lamination locations, but processes within the appropriate lamina are selectively stabilized with maturation. Process stabilization cues may be provided by other retinal neurons that have already grown stratified arbors in the forming plexiform layers. For example, the axonal terminals of the ON bipolar cells are largely distributed within the inner half of the IPL where they later contact ON ganglion cells. At this stage of axonal elaboration, as discussed later, amacrine cells that stratify in the ON sublamina have already contacted their target ganglion cells. Because amacrine cells can stratify independently of the presence of retinal ganglion cells, it is possible that these interneurons provide laminar cues for the bipolar axons. This hypothesis receives some support from observations in the zebrafish lakritz mutant that lacks retinal ganglion cells, in which there are local patches with lamination defects in the amacrine cell neurites although lamination largely occurs across the retina. In these locally perturbed regions, bipolar axon terminals are also abnormally placed (Figure 4(b)).

Photoreceptors

Photoreceptors connect to the bipolar cell dendrites once horizontal cells have already entered into synaptic partnerships with the photoreceptors (Figure 2). Apart

from early Golgi studies, relatively little is known about how individual photoreceptors terminate their axons to form a single lamina in the outer retina during development although several studies have described the maturation of their outer segments. Observations in the ferret retina showed that some photoreceptors even transiently project into the IPL during development, although the functional significance of these projections is not yet known.

Synaptogenesis in the Vertical Pathway

Synaptic connections in the vertical pathway have only recently been studied in detail by light microscopy. Early ultrastructural studies of the IPL and OPL of mammals and zebrafish suggest that ribbon synapses are formed between bipolar cells and retinal ganglion cells after conventional synapses are present (Figures 5 and 6). In rodents and zebrafish, contact between photoreceptors and bipolar cells occur about the same time as bipolar cells contact the retinal ganglion cells. However, ribbons appear late at bipolar synapses in the IPL. Detailed serial reconstruction of immature bipolar cell axon terminals in primate retina suggest that ribbons appear after clusters of vesicles are found at appositions between bipolar cell axonal processes and a neighboring process. Interestingly,

RGC activity Vesicular transporters Synaptogenesis

Highly correlated spontaneous bursting activity, waves

Spontaneous excitatory (glutamatergic) PSCs

Spontaneous inhibitory PSCs

Emergence of robust light responses