Joshua R. Sanes

Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.

Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex

Do new synapses form in the adult cortex to support experience-dependent plasticity? To address this question, we repeatedly imaged individual pyramidal neurons in the mouse barrel cortex over periods of weeks. We found that, although dendritic structure is stable, some spines appear and disappear. Spine lifetimes vary greatly: stable spines, about 50% of the population, persist for at least a month, whereas the remainder are present for a few days or less. Serial-section electron microscopy of imaged dendritic segments revealed retrospectively that spine sprouting and retraction are associated with synapse formation and elimination. Experience-dependent plasticity of cortical receptive fields was accompanied by increased synapse turnover. Our measurements suggest that sensory experience drives the formation and elimination of synapses and that these changes might underlie adaptive remodelling of neural circuits.

Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system

Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale.

Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.

We show that a gene introduced into cells of mouse embryos by a retrovirus can serve as a heritable marker for the study of cell lineage in vivo. We constructed a defective recombinant retrovirus in which the Escherichia coli beta‐galactosidase (lacZ) gene is inserted in the genome of a Muloney murine leukemia virus (M‐MuLV). Expression of lacZ was detected with a histochemical stain that can be applied to cultured cells and embryonic tissue. Infection of cultured cells showed that lacZ has no detectable deleterious effects on cell viability or growth, that the enzyme is stably expressed in the progeny of infected cells for many generations in the absence of selective pressure, and that the virus can induce lacZ in a variety of cell types. Following injection of the virus into mid‐gestation mouse embryos, clones of lacZ‐positive cells were detected in skin, skull, meninges, brain, visceral yolk sac, and amnion. We identified the cell types comprising a series of lacZ‐positive clones in the visceral yolk sac and skin to learn the lineage relationships of the labelled cells. In each tissue, we obtained evidence that several cell types have a pluripotential ancestor and that cell fate is progressively restricted as development proceeds.

INDUCTION, ASSEMBLY, MATURATION AND MAINTENANCE OF A POSTSYNAPTIC APPARATUS

Key Points The neuromuscular junction (NMJ) is one of the best models to study the induction, assembly, maturation and maintenance of a postsynaptic apparatus. The three most widely appreciated experimental advantages of the NMJ are its size, its simplicity and its accessibility. Acetylcholine receptors (AChRs) are inserted at multiple points into the embryonic myotube membrane. As the NMJ develops, receptor density underneath the nerve increases and the number of extrasynaptic receptors is very small. Several mechanisms account for this phenomenon: some AChRs redistribute in the plane of the membrane, the metabolic stability of AChRs increases after clustering, myonuclei associated with the postsynaptic membrane become transcriptionally specialized to express AChRs, and AChR transcription is suppressed in non-synaptic nuclei. This reorganization depends on chemical influences from the motor nerve. Several molecules that are responsible for postsynaptic reorganization have been identified. Their identification has led to a working model of how AChRs are clustered at the developing NMJ. In this model, the nerve releases a protein called agrin, which signals through a muscle-specific tyrosine kinase known as MuSK. MuSK then acts through an effector protein, rapsyn, to promote AChR clustering. Although this basic model has received significant experimental support, there are other factors that affect NMJ development. Neuregulin and its ErbB receptor (another tyrosine kinase) also affect AChR clustering, but the interaction of these proteins with the agrin–MuSK–rapsyn pathway is unclear. The embryonic NMJs are very different from the adult NMJs. The junction changes from a simple oval plaque to a pretzel-like set of branches, and the junctional membrane changes from a flat sheet to an invaginated surface with gutters and folds. Moreover, the composition of the basal lamina and the cytoskeletal apparatus change as the NMJ matures. Finally, a shift in AChR subunit composition leads to a change in their Ca2+ permeability, and ion- and ligand-gated channels segregate into discrete alternating domains. The mechanisms that underlie each of these transformations have only begun to be uncovered. AbstractThe postsynaptic apparatus of the skeletal neuromuscular junction, like that of other synapses, contains a high-density patch of neurotransmitter receptors that is closely associated with a variety of extracellular, transmembrane and cytoplasmic proteins that have adhesive, structural and signalling roles. The postsynaptic apparatus is organized by signals from the presynaptic nerve terminal. It changes in shape, size and molecular architecture as it matures. Once mature, it can be maintained for the life of the organism, but has the capacity for remodelling in response to altered input. The molecular and cellular mechanisms that govern each of these stages are now being elucidated by a combination of microscopic and genetic methods, allowing the neuromuscular junction to serve as a model for smaller and less-accessible central synapses.The prefrontal cortex has a vital role in effective, organized behaviour. Both functional neuroimaging in humans and electrophysiology in awake monkeys indicate that a fundamental principle of prefrontal function might be adaptive neural coding — in large regions of the prefrontal cortex, neurons adapt their properties to carry specifically information that is relevant to current concerns, producing a dense, distributed representation of related inputs, actions, rewards and other information. A model based on such adaptive coding integrates the role of the prefrontal cortex in working memory, attention and control. Adaptive coding points to new perspectives on several basic questions, including mapping of cognitive to neurophysiological functions, the influences of task content and difficulty, and the nature of frontal lobe specializations.

Defective Neuromuscular Synaptogenesis in Agrin-Deficient Mutant Mice

During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation does occur in the mutant, suggesting the existence of a second-nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.

Synaptic structure and development: The neuromuscular junction

Synaptic transmission--the process by which signals are transferred from a neuron to its target--is a fundamental function of neurons. Most neurobiologists look to the synapse to find the patterns of connectivity that account for neural specificity, the information processing that underlies behavior, and the plasticity responsible for learning. Accordingly, it is no surpris e that we are keenly interested in how synapses are formed. What is surprising is that virtually all of our current understanding of synaptogenesis derives from the study of just one synapse, the vertebrate skeletal neuromuscular junction. This synapse lies outside of the brain and does not even have a neuron as its postsynaptic element. It is the only synapse in vertebrates or invertebrates, however, whose structure and function are sufficiently well understood that the mechanisms regulating its development can be analyzed. The features of simplicity and accessibility that have enabled investigation of the mature synapse facilitate this analysis. In addition, unlike central neurons, muscles are readily reinnervated following nerve damage, allowing synaptogenesis to be studied in the adult, uncomplicated by processes such as neurogenesis that occur only in the embryo. Finally, although interneuronal synapses differ from neuromuscular junctions in important ways, our fragmentary knowledge to date encourages the belief that similar principles govern the development of both. For all of these reasons, this review is devoted to the neuromuscular junction. We begin by describing the cytological and molecular architecture of this synapse, making three main points: that chemical synapses are designed for rapid, focal transmission of information; that this task is performed by highly specialized preand postsynaptic domains that lie in precise juxtaposition across the synaptic cleft; and that many of the components forming these domains have now been isolated and characterized. We then examine the complex series of inductive interactions between nerve and muscle that regulate synaptic development. These interactions are mediated by membrane, extracellular matrix, and soluble molecules that are only now being identified. A major point is that nerve and muscle can each synthesize and assemble synaptic components on its own. In vivo, however, they do so in coordination and only at points of juxtaposition. Thus, intercellular interactions during synaptogenesis localize and refine the synaptic functions of each cell. The result is a process of synaptic maturation that proceeds to completion in a series of overlapping steps over a prolonged interval. As the signaling molecules are identified and the regulatory circuits that define these steps are unraveled, the classic histological and physiological descriptions of synaptogenesis are being reformulated in molecular and mechanistic terms.

A new nomenclature for the laminins

The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.

Skeletal and Cardiac Myopathies in Mice Lacking Utrophin and Dystrophin: A Model for Duchenne Muscular Dystrophy

Dystrophin is a cytoskeletal protein of muscle fibers; its loss in humans leads to Duchenne muscular dystrophy, an inevitably fatal wasting of skeletal and cardiac muscle. mdx mice also lack dystrophin, but are only mildly dystrophic. Utrophin, a homolog of dystrophin, is confined to the postsynaptic membrane at skeletal neuromuscular junctions and has been implicated in synaptic development. However, mice lacking utrophin show only subtle neuromuscular defects. Here, we asked whether the mild phenotypes of the two single mutants reflect compensation between the two proteins. Synaptic development was qualitatively normal in double mutants, but dystrophy was severe and closely resembled that seen in Duchenne. Thus, utrophin attenuates the effects of dystrophin deficiency, and the double mutant may provide a useful model for studies of pathogenesis and therapy.

Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus.

To analyze cell lineage in the murine cerebral cortex, we infected progenitor cells with a recombinant retrovirus, then used the retroviral gene product to identify the descendants of infected cells. Cortices were infected on E12-E14 either in vivo or following dissociation and culture. In both cases, nearly all clones contained either neurons or glia, but not both. Thus, neuronal and glial lineages appear to diverge early in cortical development. To analyze the distribution of clonally related cells in vivo, clonal boundaries were reconstructed from serial sections. Perinatally (E18-PN0), clonally related cells were radially arrayed as they migrated to the cortical plate. Thus, clonal cohorts traverse a similar radial path. Following migration (PN7-PN23), neuronal clones generally remained radially arrayed, while glial clones were variable in orientation, suggesting that these two cell types accumulate in different ways. Neuronal clones sometimes spanned the full thickness of the cortex. Thus, a single progenitor can contribute neurons to several laminae.