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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 23.7Formation of Topographic Maps and Synapses

Upon reaching a target region, growth cones must choose with which type of target cells to form connections. Typically many different types of neurons are present within a given target region, and growth cones from particular neurons will form synapses with only a subset of them. The ability of growth cones to select among different types of target neurons (or muscle cells) is referred to as cell-type specificity. In addition, in many regions of the nervous system, particularly in vertebrates, large populations of neurons of one cell type from one region of the nervous system connect to a population of another cell type in a target region. The ability of innervating neurons to maintain their spatial relationships in the field of target cells is referred to as topographic specificity. This phenomenon leads to a defined spatial relationship, or topographic map, between neurons and their targets. Although little is known about how specific neurons select their postsynaptic partners from a population of different cell types, considerable progress has been made in dissecting the molecular mechanisms regulating formation of topographic maps, particularly in the visual system of vertebrates.

Visual Stimuli Are Mapped onto the Tectum

Both electrophysiological and axon labeling experiments revealed the existence of topographic maps in the vertebrate visual system. For instance, stimulation of a specific region of the retinal field produces an electrical response in cells in defined regions of the tectum, the region of the brain to which the retinal ganglion cells project in lower vertebrates (e.g., frogs). By labeling cells in specific regions of the visual field with a dye that is transported along retinal ganglion cell axons, the map can be directly visualized in the tectum. For instance, in the chick retina, nasal axons (located near the nose) project to the posterior tectum, whereas temporal axons (located near the temple) project to the anterior tectum (Figure 23-38). Such experiments show that stimuli in the visual field are mapped in a smooth continuous manner in the tectum.

Figure 23-38. Retinotopic map in the chick.

Figure 23-38

Retinotopic map in the chick. (a) Stimulation of the visual space in the retina is directly mapped onto the tectum in the chick brain. (b) Retinal ganglion neurons in the retina elaborate (more...)

A classic series of surgical experiments in lower vertebrates produced evidence that retinal ganglion cells specifically recognize their targets in the tectum. In one set of experiments scientists removed the eye of a frog, cut the optic nerve that connects the eye to the tectum, rotated the eye 180 degrees and then placed it back into the eye capsule. When the frog was exposed to a visual stimulus, its behavioral response was directed about 180 degrees from the response elicited prior to the surgery or in control animals. The simplest interpretation of these studies, later validated using anatomical techniques, was that the retinal ganglion cells in the rotated eye connected to the position of the tectum reflecting their original position in the retinal field.

Temporal Retinal Axons Are Repelled by Posterior Tectal Membranes

The discovery of topographic maps in the visual system suggested that retinal ganglion cells in different regions of the visual field are directed to connect to different target regions in the tectum during development and that chemical differences between regions of the tectum are detected by the innervating retinal neurons. Because there are far too many neurons for each site in the tectum to express a different signal, it seemed likely that retinal axons from different regions of the retina are able to detect quantitative differences in signals. That is, the graded distribution of a chemical signal in the target region specifies the retinotopic map.

Important progress toward identifying the signals responsible for establishing the retinotopic map came from studies of chick retinal ganglion cells growing in culture. To test whether retinal neurons can distinguish between molecular signals expressed by anterior and posterior tectal cells, scientists placed retinal tissue removed from chicks in a culture containing alternating lanes of anterior and posterior tectal membranes. Although nasal axons grew over the entire plate and did not discriminate between the lanes, the temporal axons extended only along the membranes prepared from the anterior tectum (Figure 23-39a).

Figure 23-39. Effect of tectal membranes on outgrowth of chick retinal neurons.

Figure 23-39

Effect of tectal membranes on outgrowth of chick retinal neurons. (a) Temporal and nasal retinal tissues (bottom) were added to culture dishes to which stripes of anterior (A) and (more...)

By testing the response of individual growth cones to tectal membranes, scientists found that temporal growth cones collapse and fail to advance when incubated in the presence of posterior tectal membranes but are unaffected by anterior tectal membranes (Figure 23-39b). Nasal growth cones showed no response to either anterior or posterior tectal membranes. These studies led to the proposal that the graded distribution of a repellent affecting temporal growth cones plays an important role in establishing a retinotopic map.

Ephrin A Ligands Are Expressed as a Gradient along the Anteroposterior Tectal Axis

In an earlier section, we saw that ephrin B2 and its receptor, both expressed on the surface of cells, mediate reciprocal inductive interactions during angiogenesis. Another ephrin ligand/Eph receptor system has been shown to function in establishing the topographic map of retinal growth cones along the anteroposterior axis of the tectum. Although the cytosolic domain of the Eph receptors are homologous to other well-characterized receptor tyrosine kinases, there extracellular domains are novel (see Figure 23-25). One such receptor, designated EphA3, is widely expressed in the developing vertebrate brain. Since the ligands binding to the Ephs were unknown at first, these receptors were referred to as orphan receptors.

To identify the ligand(s) that binds to EphA3 (and other Ephs) scientists fused the region of the gene encoding its extracellular ligand-binding domain to a region of DNA encoding alkaline phosphatase and produced the encoded chimeric protein in cultured cells (Figure 23-40a). Researchers reasoned that the extracellular ligand-binding domain of the chimeric protein would bind tightly to ligands in tissue; the activity of the alkaline phosphatase domain then could be used as a histochemical stain to visualize the location of the bound ligands. This experimental approach detected a ligand for EphA3 that is expressed as a gradient in the tectum with high levels posteriorly and low levels anteriorly (Figure 23-40b). The receptor–alkaline phosphatase chimeric protein was also used as an affinity reagent to clone the ligand from a cDNA expression library (see Figure 20-9). This led to the isolation of the ligand, designated ephrin A2.

Figure 23-40. Identification of a graded Eph-binding signal in the tectum.

Figure 23-40

Identification of a graded Eph-binding signal in the tectum. (a) A DNA construct was engineered that encoded the extracellular domain of the Eph receptor (e.g., EphA3) fused to alkaline (more...)

A biochemical approach was used to identify another ephrin ligand (A5) that is selectively expressed in the posterior tectum. Both ephrin A2 and A5 are expressed during the period in development when the repellent activity of posterior tectal membranes on temporal growth cones is detected. Both also are linked to the membrane by a GPI anchor (see Figure 23-25). Treatment of posterior tectal membranes with phospholipase C, which cleaves the GPI anchor, inactivates their repellent activity, further evidence that ephrin A2 and A5 act as chemorepellents.

Additional evidence that ephrin A2 acts as a repellent for temporal axons came from experiments in which membranes prepared from tissue culture cells overexpressing ephrin A2 were used in a stripe assay similar to that shown in Figure 23-39a. Temporal axons preferentially migrated along stripes lacking ephrin A2, but nasal axons showed no preference. In another study, anterior tectal membranes were infected with a retrovirus whose genome encoded ephrin A2, resulting in expression of ephrin A2 in patches of the chick retina. Temporal axons avoided these patches, whereas nasal axons grew over them. To assess why different retinal axons respond differently to ephrin A2, scientists examined the distribution of one of its receptors, EphA3.

The EphA3 Receptor Is Expressed in a Nasal-Temporal Gradient in the Retina

In situ hybridization experiments demonstrated that EphA3, a receptor for ephrin A2, is expressed as a gradient in the retina, with high levels in the temporal region and low levels in the nasal region. To determine the distribution of the EphA3 receptor on retinal growth cones in the tectum, scientists generated a ligand–alkaline phosphatase fusion protein similar to the receptor fusion protein depicted in Figure 23-40a. When researchers used the ephrin A2–phosphatase fusion protein as a histological reagent, the anterior tectum was heavily stained, reflecting the high-level expression of EphA3 on temporal retinal growth cones, and the posterior tectum was lightly stained, reflecting the low-level expression of EphA3 on nasal retinal growth cones.

These data suggest that the topographic map established along the anterioposterior tectal axis reflects the graded distribution of a repellent (ephrin A2) in the target tissue and the complementary pattern of expression of the receptor for it (EphA3) in the retina (Figure 23-41). Thus temporal retinal axons, which express high levels of EphA3, are repelled by posterior tectal cells, which express high levels of ephrin A2. Conversely, since nasal retinal axons express very low levels of the EphA3 receptor, they can innervate the posterior tectum. Although these studies provide insight into why temporal axons avoid the posterior tectum, they do not explain why nasal axons do not innervate the anterior tectum. One possible explanation is that a chemoattractant selective for nasal axons is expressed preferentially in the posterior tectum.

Figure 23-41. Graded expression of ephrin A2 and its receptor, EphA3, in the chick retina and tectum.

Figure 23-41

Graded expression of ephrin A2 and its receptor, EphA3, in the chick retina and tectum. EphA3 is expressed at the highest levels on the surface of temporal retinal axons and their growth (more...)

Motor Neurons Induce Assembly of the Neuromuscular Junction

Once in the target region, the neuronal growth cone must select a specific target cell with which to form synapses. While little is known about the formation of nerve-to-nerve synapses, it is likely that the lessons learned from the detailed biochemical and genetic studies of nerve-muscle synapse, or neuromuscular junction (NMJ), formation will provide important insights into this issue. In this section, we discuss the role of an extracellular signal, agrin in NMJ formation.

The intricate cellular and molecular architecture of the NMJ is designed for rapid focal transmission of the nerve impulse to the muscle (see Figure 21-4). The highly specialized components of the synapse, including synaptic vesicles containing acetylcholine (ACh) in the presynaptic cell and acetylcholine receptors (AChR) in the postsynaptic muscle cell, must be concentrated and then assembled to lie in precise apposition from one another across the synaptic cleft. This process requires communication between the nerve and muscle cells.

Experiments on regeneration of damaged muscle and nerve, outlined in Figure 23-42, demonstrate that a specialized region of the basal lamina, which is present in the synaptic cleft, induces both nerve and muscle to form the specialized structures of the NMJ. Even if muscle regeneration is prevented, axons still return to precisely the original site on the basal lamina and form an axon terminus with synaptic vesicles. Conversely, if myofibers regenerate—but the axon does not—specialized regions of the plasma membrane enriched in AChRs form at the original synaptic site on the basal lamina.

Figure 23-42. A specialized region of the basal lamina determines the site of the neuromuscular junction.

Figure 23-42

A specialized region of the basal lamina determines the site of the neuromuscular junction. When a striated frog limb muscle and innervating motor neurons are damaged, both the muscle and (more...)

These findings led investigators to search for molecules in the basal lamina that direct where on the muscle cell a postsynaptic specialization will form. From the time of the initial contact between a neuron and a muscle cell to formation of a complete, functional synapse between them, more than 40 known molecules become concentrated specifically at the synapse. One of the earliest to become concentrated, and one of obvious physiological significance, is the AChR on the muscle-cell membrane. AChR subunits are synthesized as myoblasts fuse and differentiate into myotubes (see Chapter 14).

Prior to innervation, there are approximately 1000 AChR molecules/μm2 on the muscle surface. In the mature NMJ, the density increases to 10,000 molecules/μm2, while the density of AChR in the muscle membrane outside the NMJ falls to about 10 molecules/μm2. At least three different signaling processes control this morphological differentiation: (1) Preexisting AChR on the surface of the uninnervated muscle are induced to aggregate by agrin, a signal released from the nerve. (2) The nerve releases a factor called ARIA (Acetylcholine Receptor Inducing Activity) that stimulates transcription of AChR subunits in nuclei that underlie the developing synapse. (3) AChR released from the nerve leads to voltage changes and Ca2+ entry, which represses transcription of AChR subunits in nuclei in the muscle syncitium that do not underlie synapses. The first step in synapse induction is the release of agrin from motor neurons as they approach muscle fibers. In the next two sections, we discuss agrin and its receptor.

Agrin Induces Acetylcholine Receptor Aggregation

The localization of AChRs is easily monitored using fluorescentlabeled bungarotoxin, which directly binds to these receptors. In muscle cells cultured in the absence of motor neurons, AChRs are distributed randomly over the surface of the muscle cell. Contact with a co-cultured motor neuron results in rapid clustering of AChRs via lateral diffusion of receptor molecules in the muscle-cell plasma membrane to the site of nerve-muscle contact.

Cultured chick myotubes were used to assay the AChR–clustering activity of protein fractions of basal lamina prepared from a particularly rich source of cholinergic synapses, the electric ray Torpedo californica. From this source, investigators isolated and partially sequenced a protein they named agrin. Staining with antibodies specific for agrin showed that it is stably associated with the basal lamina at the NMJ. Furthermore, inclusion of anti-agrin antibodies in nerve-muscle co-cultures prevented the aggregation of AChRs (Figure 23-43). Agrin thus mediates aggregation of AChRs, as well as a dozen other components, at the NMJ.

Figure 23-43. Experimental demonstration that agrin, which is associated with the basal lamina, promotes aggregation of acetylcholine receptors (AChRs).

Figure 23-43

Experimental demonstration that agrin, which is associated with the basal lamina, promotes aggregation of acetylcholine receptors (AChRs). Co-cultures of nerve and muscle cells were (more...)

Sequencing of agrin cDNA revealed that it encodes a 200-kDa protein related to several known proteins, including laminin, another component of the basal lamina. The N-terminal half of the agrin molecule contains matrix-binding sites; the C-terminal half contains membrane-binding sites and is required for the AChR-clustering activity of agrin. The protein has several repeats found in other extracellular-matrix proteins or signaling molecules. Alternative splicing of agrin transcripts results in a number of agrin isoforms, some of which are inactive in AChR-clustering assays. Localization studies indicate that both muscle and motor neurons produce agrin, that motor neurons transport agrin to the nerve terminus, and that agrin from both cellular sources is localized at the neuromuscular junction. Scientists identified a neuron-specific form of agrin (formed by alternative splicing) and demonstrated that it is about 1000-fold more active in inducing AChR clustering than agrin isoforms expressed by muscle. AChRs do not aggregate and NMJs do not form in mice lacking agrin.

A Receptor Tyrosine Kinase Is Part of the Agrin Receptor

Agrin causes AChR clustering at very low concentrations (0.1–1 ppm) and exhibits saturable, high-affinity binding to muscle-cell membranes. These data and the finding that one molecule of agrin mediates clustering of ≈200 AChRs suggest that agrin does not directly bind to the AChR. Rather, it most likely functions as a signaling molecule controlling the assembly of postsynaptic specializations at the site of motor neuron contact.

A key component of the agrin receptor was identified as a muscle-specific receptor tyrosine kinase. Scientists were interested in identifying receptors for novel factors regulating muscle differentiation or survival. They reasoned that genes encoding such receptors would be upregulated in response to denervation of skeletal muscle. A single muscle-specific cDNA clone encoding a novel receptor tyrosine kinase was identified from mRNA isolated from denervated muscle. This receptor, called MuSK (Muscle Specific Kinase), is expressed at low levels in myoblasts and is upregulated with the onset of myoblast fusion and differentiation. While overall levels of this receptor decrease in mature muscle, the receptors become concentrated in postsynaptic membranes at the NMJ.

Several lines of evidence indicate that MuSK is part of a receptor complex for agrin. Knockout mice lacking MuSK exhibit a phenotype similar to the agrin knockout. While agrin induces AChR aggregation in wild-type myotubes in vitro, it does not do so in myotubes from MuSK mutants. And finally, MuSK tyrosine kinase activity is stimulated specifically by the neuronal isoform of agrin. Nevertheless, agrin does not appear to bind directly to the extracellular domain of MuSK. These and other studies indicate that additional myotube components or myotube-specific modification of MuSK is necessary to generate an active agrin receptor.

How does MuSK promote AChR aggregation? Important clues came from the analysis of rapsyn, a 43-kD protein identified in biochemical studies as copurifying with the AChR. That rapsyn can aggregate AChR was shown in gain-of-function studies. While AChRs are expressed diffusely along the surface of nonmuscle cells transfected with AChR subunit genes, cotransfection with rapsyn induced receptor clustering. Conversely, rapsyn knock-out mice fail to aggregate receptors. The precise biochemical mechanisms by which activation of MuSK promotes rapsyn-dependent aggregation of AChR is as yet unclear.

These studies suggest the following pathway for establishing the NMJ: Agrin is released from motor neuron growth cone. MuSK activates rapsyn-dependent-AChR aggregation. Many other muscle components are induced to aggregate at the synapse including the receptors for ARIA. Myotube nuclei directly underlying the receptor are induced to up-regulate transcription of synapse-specific components by ARIA released from motor neurons. Transcription of AChR genes and other postsynaptic components are repressed in nuclei in regions away from the synapse. Repression requires depolarization of the muscle membrane by activation of AChR by presynaptic release of acetylcholine from motor nerve terminals.

Figure 23-44. Signals from the motor neuron regulate the expression and localization of the acetylcholine receptor (AChR).

Figure 23-44

Signals from the motor neuron regulate the expression and localization of the acetylcholine receptor (AChR). Agrin binds to the agrin receptor, which includes MuSK and additional (more...)

SUMMARY

  •  A common feature of neuronal organization is that neuronal cell types of the same class arrayed in one field (e.g., retinal ganglion neurons in the retina) maintain their spatial relations to each other when they connect to their target cells in another field (e.g., the tectum). Such defined spatial arrangements, referred to as topographic maps, are found in the vertebrate visual system (see Figure 23-38).
  •  Topographic mapping in the visual system is regulated in part by ephrin A1, a cell-surface grow-cone repellent expressed in the tectum, and its receptor, EphA3, expressed on the growth cones of retinal neurons.
  •  Ephrin A1 and EphA3 are both expressed in a graded but complementary distribution (see Figure 23-41). Temporal retinal growth cones, which express high levels of Eph3 receptor, are repelled by the high levels of ephrin A1 produced in the posterior tectum. Conversely, nasal retinal growth cones, which express low levels of Eph3 receptor, are less sensitive to the repelling activity of ephrin 1 in the posterior tectum, permitting them to form connections with target cells in this region.
  •  A specialized chemical synapse, the neuromuscular junction, is established between a motor neuron and its target muscle. Formation of this junction requires the concentration, assembly, and precise alignment of numerous components in both the innervating nerve and the muscle cell.
  •  Agrin, is released from the motor neuron and mediates clustering of acetylcholine receptors and other components in the neuromuscular junction. MuSK is a component of the agrin receptor on muscle membranes. Rapsyn is a cytosolic protein that binds to the AChR and mediates clustering.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21742

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