Introduction

Under normal physiological conditions, neurons do not come in direct contact with blood. The blood–brain barrier, consisting of astrocyte end feet, extracellular matrix, and endothelial cells, forms an elaborate meshwork that surrounds blood vessels and regulates the selective passage of blood elements and nutrients to the neurons. When an artery in the brain ruptures, blood envelopes cells in the surrounding tissue, upsets the blood supply provided by the injured vessel and disturbs the delicate chemical equilibrium essential for neurons to function. This is called hemorrhagic stroke and accounts for approximately 20% of all strokes.

Hemorrhagic stroke has been less investigated than ischemic stroke although it represents a significant clinical problem. Direct tissue destruction, tissue compression around the hematoma, and an inflammatory response lead to neuronal injury and neurological deficits after hemorrhagic strokes. The size of the hematoma has a direct relationship with the clinical outcome. The hematoma causes mass effect and compresses the surrounding tissue, contributing to the neuronal death at the margin of the hematoma and in the penumbral region around the hematoma. Decreasing the space-occupying effect by aspiration of the hematoma and decreasing inflammation ameliorate the neurological deficits after hemorrhagic stroke.

A number of experimental cerebral hemorrhagic models have been developed to study the mechanisms underlying cerebral bleeding and resulting pathophysiology. The knowledge gained has helped in identifying many factors that contribute to rupture of an artery or an aneurysm.

Introduction

Analysis of the brain tissue depends on the experimental setup and question, and needs to be decided by each investigator. However, one should bear in mind that the health of animals as well as the diet they eat will inevitably affect the results obtained. Even though it is commonly thought to be so, the experiment does not start from the day the brains are dissected out and subjected to analysis but from the way animals are handled, fed, and cared for.

Fixation of brain tissue

Fixation is needed to stop degradation of the tissue and to preserve both structure and tissue antigens for analysis. Chemicals used for fixation are compounds that form cross-linking bonds between the components of the tissue and thereby literally fix/preserve them in the state they existed during life. The more cross-linking in the fixative, the more it can preserve the structural morphology of the tissue. The most commonly used fixatives in the order of their cross-linking properties are: glutaraldehyde, formaldehyde, paraformaldehyde, and p-benzoquinone. In addition, different alcohols (acetone, methanol) can be used as fixatives, but they dissolve lipids and therefore do not preserve structure as well.

For electron microscopic analysis of the brain, 1–2% glutaraldehyde is the preferred fixative. For immunocytochemical demonstration of tissue antigens at light microscopic level, 2–4% paraformaldehyde or 0.4% p-benzoquinone are the best alternatives.

Introduction

Once various cell types have migrated to their final location, they start synthesizing neuro–transmitters and extend neurites. At that stage, they are ready to begin forming synaptic connections with other retinal neurons. We have already seen in Chapter 6 that neurotransmitters are present before the formation of functional synapses, before electrical activity can be detected, suggesting that they play a trophic role during retinal development. However, retinal visual processing, the conversion of light into electrical signals and the relaying of these signals to the visual centres of the brain, cannot occur until neurons have established synaptic contacts with each other.

The first part of this chapter describes the formation of synaptic connections in the various retinal layers. In the last decade, important issues regarding the establishment of synaptic connections have been resolved thanks to the advent of genetic engineering and to the development of powerful specific cellular or subcellular markers, some of which are reviewed here. We put a particular emphasis on the formation of synapses between photoreceptors and second-order neurons because photoreceptors are involved in many various types of hereditary retinal degenerations. We review studies using transgenic mouse models because they provide invaluable knowledge about factors influencing photoreceptor synaptogenesis (Farber and Danciger, 1997). Understanding factors that affect synapse formation between photoreceptors and retinal neurons is crucial for reaching better insights into these devastating diseases that often lead to blindness. We will also discuss briefly the formation of gap junctions.

Introduction

The optic nerve is the anatomical pathway through which visual information received in the retina is conveyed along the axons of retinal ganglion cells (RGCs) to central visual targets for processing. In terms of its cellular organization, the optic nerve is relatively simple compared with other white matter tracts in the CNS. Unlike most CNS axon pathways, which typically contain ascending and descending axons from multiple neuronal populations, axons within the optic nerve all originate from RGCs in the eye, and all project in the same direction away from the retina towards the brain. There are no neurons in the optic nerve, and all resident cell nuclei belong to optic nerve glial cells. Given these organizational features, the developing optic nerve is an attractive experimental system and, not surprisingly, has been widely used in studies of axon guidance, glial differentiation, glial migration and myelination. Similarly, the adult optic nerve has also served extremely well as a model for studies of axonal transport and axon regeneration. This chapter describes the developmental mechanisms governing major aspects of optic nerve formation such as the determination of optic stalk cell fate, axon guidance and glia migration. The aim is to highlight our current understanding of these developmental processes, which at a basic level are fundamental to development of all regions of the nervous system.

Phases of optic nerve development

In considering optic nerve development, it is useful to conceptually divide the process into three phases.

Vision is undoubtedly our most ‘cherished’ sense, and blindness the most tragic loss in perceiving the world around us. Visual perception begins in the eye, of which the retina is the most important component for interpreting visual signals, including colour, shape and movement. The retina is an ocular extension of the brain specialized in receiving and processing light and images. Although it is merely a few 100 micrometres thick and contains only seven cell types, the retina performs very sophisticated visual processing. Ultimately, it sends ALL information about the outside world to visual centres of the brain via the optic nerve in the form of coded electrical impulses. Understanding how the retina is organized and how it functions is thus of fundamental importance for understanding the entire visual system. It is therefore not surprising that the retina has been the focus of attention of many scientists since the late nineteenth century, when Cajal, in 1893, provided the first account of the anatomical organization of the vertebrate retina.

Although our knowledge of how the retina is organized and functions in adult organisms is absolutely essential, understanding how it is assembled during development is no less important. Indeed, when normal development is impaired, irreversible damage can result, in some cases even blindness. Moreover, understanding how the retina develops is attractive not only to developmental neuroscientists interested in vision, but to all neuroscientists interested in development, because the retina is ‘an approachable part of the brain’, and developmental processes required to build this exquisitely organized system, with well-defined layers and a limited number of cell types, are ultimately relevant to all other parts of the central nervous system.

Introduction

Müller cells, the principal glia of vertebrate retinas, are radial glia that span the entire depth of the retina. The distal processes of Müller cells form the external limiting membrane of the retina, while their ‘endfeet’ form the inner limiting membrane. Müller cell processes surround neuronal cell bodies in the nuclear layers and contact synapses in the plexiform layers (Newman and Reichenbach, 1996). Müller cells play a major role in regulating extracellular K+ and pH (Newman et al., 1984; Karwoski et al., 1989; Kusaka and Puro, 1997), in neurotransmitter uptake (Pow, 2001) and in glutamine synthesis (Riepe, 1977, 1978; Germer et al., 1997a; Prada et al., 1998), functions performed by astrocytes in other regions of the central nervous system. Müller cells also have some similarities to oligodendrocytes; although they do not form myelin, Müller cell processes wrap the axons of retinal ganglion cells (Holländer et al., 1991; Stone et al., 1995). In addition, intercellular Ca2+ waves have been observed among Müller cells (Newman and Zahs, 1997). These waves are increases in glial cytosolic Ca2+ that propagate away from the site of initial activation. The arrival of Ca2+ waves in retinal glia is correlated with modulation of the light-evoked activity of neighbouring retinal ganglion cells (Newman and Zahs, 1998). Modulation of retinal ganglion cell activity has been shown to be mediated by a variety of factors released by Müller cells, including purine nucleotides (Newman, 2003) and d-serine, a co-agonist at the N-methyl-d–aspartate (NMDA) type of glutamate receptor (Stevens et al., 2003).

Introduction

Retinal neuron arbors are organized in relation to three central functions. (1) Outgrowth is regulated in the lateral dimension to delimit receptive-field size, a property linked to spatial acuity. (2) Interactions between individual neuronal subtypes are coordinated with respect to neuritic overlap to promote complete coverage, or tiling, of the retina, thus assuring that distinct functions have representation over the entire area of the retina (see Chapter 10). (3) Interactions between pre- and postsynaptic partners are organized in the vertical dimension such that functionally discrete circuits are physically isolated within the synaptic neuropil. For instance, during development of the inner plexiform layer (IPL) connections between subsets of bipolar, amacrine and retinal ganglion cells come to be arranged in a laminar fashion, sometimes occupying single strata within a multilayered array of concentric circuits (Figure 12.1).

In this chapter the current state of understanding regarding the structural development of retinal neuron arbors is discussed: from mechanisms that impact individual neuronal morphologies to those that orchestrate interactions between synaptic partners. In the first section, issues concerning initial neurite extension are discussed. These include establishing cellular polarity and compartmentalization of neurites into the axon and dendrites. Section two focuses on the establishment of dendritic territory and interactions that influence receptive-field size. The last section deals with the process of sublamination, whereby individual neuritic arbors resolve into monostratified, multistratified, or diffuse (non-stratified) configurations within the IPL.

Introduction

Many distinct processes occur during the course of retinal development. These range from regulation of mitosis and cell fate specification to axon outgrowth and targeting, dendritogenesis and terminal differentiation of different cell types. Since all of these events require changes in gene expression, it follows that global analysis of changes in transcription during development should reveal the identity of many of the genes that mediate these processes. This has been the logic underlying genomic studies of the developing retina, which have so far been undertaken by a number of groups.

The retina has many features that make it well suited to genomic studies. In both invertebrates and vertebrates, the major cell subtypes in the retina are easily distinguished by both molecular and morphological criteria. Compared with other parts of the nervous system, the number of distinct retinal cell subtypes is quite limited and, in both rodents and flies, photoreceptors make up the majority of retinal cells. The birth order of each major cell type is known, and in vertebrates these generation times are distinct and only partially overlapping. Cell types are readily identified by spatial position, which renders in situ hybridization-based verification of primary expression data relatively straightforward. Interpretation of expression data in model organisms is also aided by previous work that has already identified large numbers of genes that are selectively expressed in specific cell types of the mature and differentiating retina. Finally, a wealth of mutations that disrupt different aspects of retinal development are available.

Vision begins at the retina, a light-sensitive tissue at the back of the eye that comprises highly organized, laminated networks of nerve cells. Investigating the mechanisms of retinal development is fundamentally important to gaining a basic knowledge of how vision is established. In this book, we present the sequence of developmental events and the mechanisms involved in shaping the structure and function of the vertebrate retina.

Formation of the eye

The eye is derived from three types of tissue during embryogenesis: the neural ectoderm gives rise to the retina and the retinal pigment epithelium (RPE), the mesoderm produces the cornea and sclera, and the lens originates from the surface ectoderm (epithelium). During embryogenesis (Figure 1.1), the eyes develop as a consequence of interactions between the surface ectoderm and the optic vesicles, evaginations of the diencephalon (forebrain). These optic vesicles are connected to the developing central nervous system by a stalk that later becomes the optic nerve. When the optic vesicles contact the ectoderm, inductive events take place to cause the epithelium to form a lens placode. The lens placode then invaginates, pinches off eventually and becomes the lens. During these events, the optic vesicle folds inwards and forms a bilayered cup, the optic cup. The outer layer of the optic cup differentiates into the RPE whereas the inner layer differentiates into the retina. The iris and ciliary body develop from the peripheral edges of the retina.

The editors have assembled an impressive authorship to produce this book on development of the retina. There are several reasons why this is timely. Over the last decade there have been rapid advances in our understanding of the mechanisms involved in formation of the eye and determination of the fate of cells. This has been driven by an explosion of laboratory techniques that have allowed the study of gene expression and characterization of cell and tissue behaviour.

As a consequence there is increasing knowledge of what determines cell function, and of the behavioural relationship between cells. This has resulted in an understanding of genetically determined disease in humans. Many genes' products have been identified during development because they are highly expressed and mutations in these genes have been identified as being responsible for developmental abnormalities in man. Some of these genes express at low levels in adult life fulfilling a house-keeping function, and mutations in these have also been identified as giving rise to progressive retinal degeneration.

Findings from studies of development are of crucial importance to the current attempts to devise biological treatment of retinal diseases. There is ample evidence that growth factors delay cell death due to apoptosis in genetically determined retinal dystrophies in animals, and therapeutic trials in man have been initiated. There is still some doubt as to which agent may be the most appropriate to achieve suppression of apoptosis.

Introduction

One of the most striking aspects of the architecture of the retina is its highly organized structure. Retinal neurons are positioned in three different layers, at different depths. Usually, all cells of a particular type are found in just one of those layers. When the spatial distribution of one type of cells within a layer can be observed, the cell bodies are arranged in a semi-regular pattern, rather than distributed randomly across the surface (Figure 10.1). These patterns are often termed ‘retinal mosaics’, due to the way that the cell bodies and dendrites of a type of neuron tend to tile the retina.

This regular arrangement of cells is thought to ensure that the visual field is evenly sampled, avoiding any perceptual blind spots in the visual field. The retina is assembled as an array of functional units, each detecting, processing and conveying to the brain information about a limited portion of the visual scene. The presence of regular arrays of neurons of the same type has long been considered a consequence of this functional design. However, recent studies have shown that retinal mosaics form early in development, before all the elements of the functional units have been born. This chapter reviews our present knowledge of the various mechanisms by which retinal mosaics emerge during development, and summarizes the mathematical techniques used to analyse mosaics.

Introduction

Vertebrate eyes originate from a single field of neuroectodermal cells in the anterior region of the neural plate called the eye field (sometimes referred to as the eye anlage, eye primordia or presumptive eye). The origins of the eye field can be traced back to the 32-cell-stage blastula in which a subset of blastomeres is competent, but not yet committed, to form retina. This chapter begins with a discussion of retinal competence and the maternal molecules and cell–cell interactions that take place in and bias early blastomeres toward a retinal fate. Transplantation experiments have shown that the entire presumptive neural plate of midgastrula embryos can form retina, demonstrating the remarkable coordination of neural development with eye formation. Neural induction and the neural patterning events critical for defining where the eye field forms in the developing nervous system will be addressed. Cultured amphibian anterior neural plates form eyes demonstrating that the eye field is specified (committed to form the eye) by the neural plate stage. A conserved set of transcription factors collectively referred to as eye field transcription factors are required for normal eye formation and are expressed in the eye field of the neural plate stage embryo. These genes and their functional interactions, which are required for and under some circumstances sufficient to drive eye field and eye formation will be described. A description of how the single vertebrate eye field separates to form the eye primordia that eventually give rise to the two eyes concludes this chapter.