Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy.

Myotonic dystrophy (DM) is an autosomal dominant disorder resulting from the expansion of a CTG repeat in the 3' untranslated region of a putative protein kinase (DMPK). To elucidate the role of DMPK in DM pathogenesis we have developed Dmpk deficient (Dmpk-/-) mice. Dmpk-/-mice develop a late-onset, progressive skeletal myopathy that shares some pathological features with DM. Muscles from mature mice show variation in fibre size, increased fibre degeneration and fibrosis. Adult Dmpk-/-mice show ultrastructural changes in muscle and a 50% decrease in force generation compared to young mice. Our results indicate that DMPK may be necessary for the maintenance of skeletal muscle structure and function and suggest that a decrease in DMPK levels may contribute to DM pathology.

Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector.

Limb-girdle muscular dystrophies 2C-F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.

Functional gap junctions in the schwann cell myelin sheath.

The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt-Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32-null mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway.

Loss of correlated motor neuron activity during synaptic competition at developing neuromuscular synapses.

During late stages of neural development, synaptic circuitry is edited by neural activity. At neuromuscular synapses, the transition from multiple to single innervation is modulated by the relative pattern of activity among inputs competing for innervation of the same muscle fiber. While experimental perturbations of activity result in marked changes in the timing of neuromuscular synaptic competition, little is known about the patterns of activity present during normal development. Here, we report the temporal patterning of motor unit activity in the soleus muscle of awake, behaving neonatal mice, and that patterning is modulated by gap-junctional coupling. Our work suggests that neuromuscular synaptic competition is modulated by surprisingly low levels of activity and may be triggered by the disappearance of temporally correlated activity among inputs competing for innervation of the same muscle fiber.

Gradual loss of synaptic cartels precedes axon withdrawal at developing neuromuscular junctions.

We have studied the spatial deployment of synapses arising from different axons that converge on the same developing neuromuscular junctions. Labeling the competing synaptic "cartels" with different dyes in mouse muscle showed that, perinatally, each axon adds similar terminal areas, whereas later, areas occupied by the competing cartels diverged by gradual elimination of one axon's synapses and ongoing addition of synaptic area by the other. Activity-dependent labeling of synapses capable of vesicle recycling in snake muscle also revealed a gradual change in territories occupied by competing inputs, implying that an axon maintained some functional synapses even as others in its cartel were being eliminated. Thus the process of synapse elimination is gradual, with loss of one viable synapse after another, until an axon is left with no synaptic territory and withdraws.

Disruption of Trkb-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions.

Neurotrophins and tyrosine receptor kinase (Trk) receptors are expressed in skeletal muscle, but it is unclear what functional role Trk-mediated signaling plays during postnatal life. Full-length TrkB (trkB.FL) as well as truncated TrkB (trkB.t1) were found to be localized primarily to the postsynaptic acetylcholine receptor- (AChR-) rich membrane at neuromuscular junctions. In vivo, dominant-negative manipulation of TrkB signaling using adenovirus to overexpress trkB.t1 in mouse sternomastoid muscle fibers resulted in the disassembly of postsynaptic AChR clusters at neuromuscular junctions, similar to that observed in mutant trkB+/- mice. When TrkB-mediated signaling was disrupted in cultured myotubes in the absence of motor nerve terminals and Schwann cells, agrin-induced AChR clusters were also disassembled. These results demonstrate a novel role for neurotrophin signaling through TrkB receptors on muscle fibers in the ongoing maintenance of postsynaptic AChR regions.

Dynamic roles at the neuromuscular junction. Schwann cells.

Recent work shows that the non-myelinating 'terminal' Schwann cells that cap neuromuscular junctions play an important role in synaptic maintenance and repair.

Nerve injury induces gap junctional coupling among axotomized adult motor neurons.

Neonatal spinal motor neurons are electrically and dye-coupled by gap junctions, but coupling is transient and disappears rapidly after birth. Here we report that adult motor neurons become recoupled by gap junctions after peripheral nerve injury. One and 4-6 weeks after nerve cut, clusters of dye-coupled motor neurons were observed among axotomized, but not control, lumbar spinal motor neurons in adult cats. Electrical coupling was not apparent, probably because of the electrotonic distance between dendrodendritic gap junctions and the somatic recording location. Analyses of gap junction protein expression in cat and rat showed that the repertoire of connexins expressed by normal adult motor neurons, Cx36, Cx37, Cx40, Cx43, and Cx45, was unchanged after axotomy. Our results suggest that the reestablishment of gap junctional coupling among axotomized adult motor neurons may occur by modulation of existing gap junction proteins that are constitutively expressed by motor neurons. After injury, interneuronal gap junctional coupling may mediate signaling that maintains the viability of axotomized motor neurons until synaptic connections are reestablished within their targets.

Disparity in neurotransmitter release probability among competing inputs during neuromuscular synapse elimination.

Competition among the several motor axons transiently innervating neonatal muscle fibers results in an increasing disparity in the quantal content and synaptic territory of each competitor, culminating in the permanent loss of all but one axon from neuromuscular junctions. We asked whether differences in the probability of neurotransmitter release also contribute to the increasing disparity in quantal content among competing inputs, and when in the process of competition changes in release probability become apparent. To address these questions, intracellular recordings were made from dually innervated neonatal mouse soleus muscle fibers, and quantal content and paired-pulse facilitation were evaluated for each input. At short interpulse intervals, paired-pulse facilitation was significantly higher for the weaker input with the smaller quantal content than the stronger input with the larger quantal content. Because neurotransmitter release probability across all release sites is inversely related to the extent of facilitation observed after paired-pulse stimulation, this result suggests that release probability is lower for weak compared with strong inputs innervating the same junction. A disparity in the probability of neurotransmitter release thus contributes to the disparity in quantal content that occurs during synaptic competition. Together, this work suggests that an input incapable of sustaining a high release probability may be at a competitive disadvantage for synaptic maintenance.

Gap junctional coupling and patterns of connexin expression among neonatal rat lumbar spinal motor neurons.

Interneuronal gap junctional coupling is a hallmark of neural development whose functional significance is poorly understood. We have characterized the extent of electrical coupling and dye coupling and patterns of gap junction protein expression in lumbar spinal motor neurons of neonatal rats. Intracellular recordings showed that neonatal motor neurons are transiently electrically coupled and that electrical coupling is reversibly abolished by halothane, a gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across most gap junctions, into single motor neurons resulted in clusters of many labeled motor neurons at postnatal day 0 (P0)-P2, and single labeled motor neurons after P7. The compact distribution of dye-labeled motor neurons suggested that, after birth, gap junctional coupling is spatially restricted. RT-PCR, in situ hybridization, and immunostaining showed that motor neurons express five connexins, Cx36, Cx37, Cx40, Cx43, and Cx45, a repertoire distinct from that expressed by other neurons or glia. Although all five connexins are widely expressed among motor neurons in embryonic and neonatal life, Cx36, Cx37, and Cx43 continue to be expressed in many adult motor neurons, and expression of Cx45, and in particular Cx40, decreases after birth. The disappearance of electrical and dye coupling despite the persistent expression of several gap junction proteins suggests that gap junctional communication among motor neurons may be modulated by mechanisms that affect gap junction assembly, permeability, or open state.

In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes.

Adenoviral vectors have been shown to effect efficient somatic gene transfer in skeletal muscle and thus offer potential for the development of therapy for Duchenne muscular dystrophy (DMD). Efficient transfer of recombinant genes has been demonstrated in skeletal muscle using recombinant adenoviruses deleted of E1. Application of this vector system to the treatment of DMD is limited by the vector immunogenicity, as well as by size constraints for insertion of recombinant genes, precluding the incorporation of a full-length dystrophin minigene construct. We describe in this study the use of helper adenovirus to generate a recombinant vector deleted of all viral open reading frames and containing a full-length dystrophin minigene. We show that this deleted vector (delta vector) is capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Our modification of adenoviral vector technology may be useful for the development of gene therapies for DMD and other diseases.

The role of the gap junction protein connexin32 in the pathogenesis of X-linked Charcot-Marie-Tooth disease.

Mutations in the gene encoding the gap junction protein connexin32 (Cx32; beta 1) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX), a common form of inherited demyelinating neuropathy. Cx32 is localized to the paranodes and incisures of myelinating Schwann cells, and probably participates in the formation of gap junctions at these locations, thereby allowing the diffusion of ions and small molecules directly across the myelin sheath. In transfected cells different CMTX mutations have different effects on the ability of the mutant protein to form functional gap junctions; some mutant proteins cannot be detected within the cell, other mutant proteins accumulate within the cell but do not reach the cell membrane, while other mutants reach the cell membrane and some of these form functional gap junctions. In transgenic mice two mutants, R142W and 175 frameshift, have similar effects on protein trafficking as in transfected cells: the R142W mutant protein remains in the perinuclear region and does not reach the paranodes or incisures, and the 175 frameshift protein cannot be detected. Thus, different CMTX mutations have different effects on Cx32 protein, and these differences may help to explain the phenotypic differences seen in CMTX kindreds.

Activity-dependent editing of neuromuscular synaptic connections.

Work over the past four decades has suggested that neural activity edits synaptic connections throughout the developing nervous system. Synaptic editing is shaped in large part by competitive interactions among different inputs innervating the same target cell that profoundly influence synaptic strength and structure. While competition plays out among presynaptic inputs that anterogradely influence their targets, postsynaptic target cells also modulate competition, in part through retrograde interactions that modulate presynaptic neurotransmitter release. One of the most useful synapses for studying how neural activity mediates synaptic editing is the connections between spinal motor neurons and skeletal muscle fibers, called neuromuscular junctions. Here we review current ideas about the role of activity in editing neuromuscular synaptic connections. The mechanisms by which activity mediates synaptic competition at these peripheral synapses are relevant to understanding how neural circuits in the central nervous system are continually altered by experience throughout life.

In vivo approaches to neuromuscular structure and function.

Approaches that permit direct observation and manipulation of skeletal muscle and its innervation in living animals will continue to contribute to our understanding of neural influences on muscle function in developing and mature animals. Understanding how motor neurons interact with each other, with supporting cells such as Schwann cells, and with their target muscle fibers are fundamental issues in neuroscience, as similar mechanisms are likely to underlie the formation and plasticity of synaptic connections in the less easily accessible central nervous system.

Gap junctional communication among developing and injured motor neurons.

The functional significance of gap junctional coupling among neurons is poorly understood. We are studying gap junctions among spinal motor neurons as a model for understanding roles of interneuronal gap junctional communication during development and after injury. Electrical and dye coupling is widespread among neonatal motor neurons but is transient, disappearing by the end of the first postnatal week. Reverse transcription polymerase chain reaction (RT-PCR) analysis, in situ hybridization and immunohistochemistry show that five rodent connexins, Cx36, Cx37, Cx40, Cx43 and Cx45, are expressed by developing motor neurons. These gap junction proteins remain expressed in some motor neurons through adult life, with the exception of Cx40, whose expression appears to decrease shortly after birth. After nerve injury in adult animals, motor neurons once again become dye coupled, and this appears to occur without dramatic changes in connexin expression. The transient gap junctional coupling present among developing motor neurons, which is re-capitulated after axotomy, may mediate electrical or biochemical signaling that shapes neuronal function.

Nip and tuck at the neuromuscular junction: a role for proteases in developmental synapse elimination.

During late embryonic and early postnatal development, synaptic connections are extensively modified so that some functional connections are weakened and eliminated from a neural circuit while others are strengthened and maintained. The mechanisms that underlie synapse elimination are beginning to be understood from studies of the neuromuscular junction. A recent paper provides some intriguing insights into the role proteases may play in the developmental disassembly of neuromuscular synapses.

Long-term synapse loss induced by focal blockade of postsynaptic receptors.

Focal application in vivo of alpha-bungarotoxin to block neurotransmission in a small region of a neuromuscular junction causes long-lasting synapse elimination at that site. In contrast, blockade of neurotransmission throughout a junction does not cause synapse elimination. These and related experiments indicate that active synaptic sites can destabilize inactive synapses in their vicinity.

The organization and development of compartmentalized innervation in rat extensor digitorum longus muscle.

1. We have examined the innervation of the rat extensor digitorum longus (EDL) muscle by the two extramuscular branches formed from the bifurcation of its muscle nerve. Observations of muscle contractions, recordings of end-plate potentials, and glycogen depletion of young adult muscles show that each branch innervates a separate region or 'compartment' in the muscle. The branch entering the muscle nearer the knee (the K branch) innervates fibres in the anteromedial half of the muscle whereas the branch entering closer to the foot (the F branch) innervates fibres located posterolaterally. Individual EDL motoneurones project either into the K or the F branch and therefore innervate fibres located in one compartment. The boundary between the compartments is usually sharply delineated. No obvious anatomical feature exists within the muscle which would explain the division of the muscle into two distinct regions. 2. The presence of a segmentotopic projection from the spinal cord to the muscle was investigated to evaluate its possible contribution to the compartmental pattern. The most posterior neurones of the EDL motor pool were found to project more frequently to the posterolateral F compartment; similarly, the most anterior neurones most frequently project to the anteromedial K compartment. However, each compartment is innervated by both anteriorly and posteriorly located motoneurones. The segmentotopic projection is too weak to explain the presence of neuromuscular compartments. 3. The post-natal period of synapse elimination appears to play at best a minor role in setting up the compartmentalized innervation. Glycogen depletion and intracellular recording in 1-2-day-old muscles show that each nerve branch innervates fibres in the same region of the muscle as in the adult. Most of the fibres in each compartment are polyneuronally innervated by axons in their own particular nerve branch, although fibres located near the boundary between the two compartments are innervated by axons from both nerve branches. This convergent innervation from the two branches disappears in concert with the elimination of polyneuronal innervation throughout the muscle. A random elimination of these convergent inputs appears adequate to explain the final compartmental pattern. 4. Our findings suggest that the compartmental pattern is primarily the consequence of te segregation of EDL motoneurones into two nerve branches which are directed into separate regions of the muscle.

Synaptic rearrangements and alterations in motor unit properties in neonatal rat extensor digitorum longus muscle.

1. We have used in vitro intracellular recordings and measurements of the contractile properties of single motor units to examine the changes in muscle innervation occurring during the post-natal development of a fast-twitch muscle in the hindlimb of the rat, the extensor digitorum longus (EDL). 2. Intracellular recordings of end-plate potentials evoked in response to graded stimulation of the nerve supply to the muscle indicate that during the first day after birth, each muscle fibre receives synaptic input from at least two motoneurones and that some muscle fibres receive as many as six such inputs. With subsequent development, most of this polyneuronal innervation is eliminated: the first singly innervated fibres are encountered on day 3; by day 18 fewer than 5% of the fibres remain polyneuronally innervated. These results show that there are quantitative differences in post-natal synapse elimination in EDL compared to its well-studied counterpart, the soleus. Although the great majority of fibres in both muscles become singly innervated at about 18 days, the first singly innervated fibres appear at least a week earlier in the EDL. None the less, synapses are lost from EDL at about half the rate they are lost from soleus. 3. The number of motor units, determined by counting the number of twitch increments produced by graded stimulation of ventral root filaments teased to contain only a few EDL motor axons, remains unchanged from an average of forty-one from post-natal day 1 to day 17. In addition, the number of muscle fibres counted in muscle cross-sections stained with an anti-myosin antibody increases less than 10% from birth to adulthood. Therefore, synapse elimination in EDL occurs with a largely constant population of muscle fibres as well as motoneurones. 4. Measurements of tensions generated by single motor units indicate that the average size of a motor unit declines from 6.8% of the muscle fibres at day 1 to 2.3% at 17 days. This result indicates that each motoneurone, on average, comes to innervate threefold fewer muscle fibres. Motor units derived from each of the spinal segments innervating the muscle undergo equivalent reductions in motor unit size, indicating that there is no segmental disproportion to synapse elimination in this muscle. At all ages, there is a large diversity of motor unit sizes in the muscle. Synapse elimination therefore appears to maintain rather than decrease this diversity.(ABSTRACT TRUNCATED AT 400 WORDS)