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R J Balice-Gordon

Publications and source records attributed to R J Balice-Gordon.

30 records · Page 2Linked to original sources
PubMed1996

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.

Adenoviridae↗
PubMed1996

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

In vivo observations of pre- and postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions.

Synaptic rearrangements in developing muscle were studied by visualizing individual neuromuscular junctions in the sternomastoid muscle of living neonatal mice as they underwent the transition from multiple to single innervation. Vital staining of ACh receptors (AChRs) with rhodamine-conjugated alpha-bungarotoxin showed that while junctions were still multiply innervated (usually by two motor axons), regions of the postsynaptic membrane within each junction became depleted of receptors. Usually, several small postsynaptic areas lost AChRs in succession. In these areas, AChRs already in the membrane rapidly disappeared compared to a low level of receptor turnover elsewhere in the junction. Moreover, there was no evidence of new AChRs being inserted into these areas. Within each postsynaptic area undergoing AChR depletion, the intensity of receptor staining decreased gradually over 1-2 d. In some junctions, it appeared that AChRs were migrating away from areas being depleted of receptors. The depletion of AChRs from some sites in combination with the spreading apart of the entire receptor-rich area due to muscle fiber growth accounts for the transformation from plaque-like to branched receptor distributions at developing neuromuscular junctions. Vital staining of presynaptic motor nerve terminals at junctions whose postsynaptic AChRs were also stained showed that motor nerve terminals were lost from the same areas that were depleted of receptors postsynaptically. Postsynaptic areas began to be depleted of AChRs before there was any obvious loss of membrane or intracellular staining in the overlying nerve terminal. Only when a single innervating axon remained at a junction did loss of motor nerve terminals and underlying AChRs largely cease. That former synaptic areas could at later times be identified as uninnervated regions within a junction indicates that synapse elimination during development leaves an indelible mark on synaptic structure. These observations suggest that the withdrawal of a motor axon from a neuromuscular junction occurs as a consequence of the stepwise elimination of all of its synapses with that muscle fiber. These results also suggest that an important aspect of synaptic competition leading to axon withdrawal is the precocious loss of AChRs beneath the nerve terminals of the axon that will be eliminated. A similar early loss of AChRs beneath one axon's synapses has been shown to occur during synapse elimination in reinnervated adult muscle (Rich and Lichtman, 1989a).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

In vivo visualization of the growth of pre- and postsynaptic elements of neuromuscular junctions in the mouse.

In order to study how neuromuscular junctions grow, we have repeatedly viewed the same junctions in mouse sternomastoid muscles at monthly intervals from 2 weeks to 18 months of age. Motor nerve terminals were stained with the nontoxic fluorescent dye 4-Di-2-ASP (Magrassi et al., 1987), and postsynaptic ACh receptors were labeled with fluorescently tagged alpha-bungarotoxin. Neuromuscular junctions grew primarily by expansion of existing motor nerve terminal and postsynaptic receptor regions without the addition or loss of synaptic areas. The expansion of pre- and postsynaptic specializations was precisely matched, suggesting that as neuromuscular junctions grow, the opposing specializations enlarge simultaneously. Each neuromuscular junction grew in length and width at the same rate that muscle fibers enlarged in those 2 dimensions, suggesting that junctional growth might be a mechanical consequence of muscle fiber growth. Repeated visualization of ACh receptors over time showed that previously labeled receptors spread apart in the membrane occupying a progressively larger area as muscle fibers grew. At the same time, new receptors were intercalated throughout the enlarged postsynaptic area. Thus, the growth of postsynaptic regions appears to be directly related to the expansion of the muscle fiber membrane as muscle fibers grow. The maintained alignment between growing motor nerve terminals and postsynaptic regions suggests that nerve terminal growth may be a consequence of its adhesion to growing postsynaptic specializations. This conclusion is supported by the coextensive stretching of motor nerve terminals and postsynaptic regions when muscle fibers are stretched. Thus, the growth of motor nerve terminals is coupled to the growth of postsynaptic regions, and the growth of the postsynaptic regions is in turn coupled to the growth of muscle fibers. In this way, the branching pattern of neuromuscular junctions may be stably maintained despite ongoing enlargement of synaptic area.

Animals↗
PubMed1990

Neuromuscular junctions shrink and expand as muscle fiber size is manipulated: in vivo observations in the androgen-sensitive bulbocavernosus muscle of mice.

Neuromuscular synapses in an androgen-sensitive muscle of sexually mature male mice were repeatedly observed over several-month intervals in normal animals and in animals in which testosterone levels were manipulated. In normal bulbocavernosus muscles, pre- and postsynaptic regions of neuromuscular junctions enlarge as muscle fibers grow. After castration, junctional area decreased in parallel with muscle fiber atrophy. When testosterone was resupplied to castrated animals, junctions that previously decreased in size then enlarged in parallel with muscle fiber hypertrophy. Surprisingly, these size changes occurred without loss or addition of motor nerve terminal branches or acetylcholine (ACh) receptor regions. Rather, each nerve terminal branch and underlying receptor region became smaller following castration and reenlarged following testosterone treatment. Several lines of evidence argued that the size changes observed after castration and testosterone treatment were secondary to shrinkage and stretching of the postsynaptic muscle fiber membrane. Following castration, the spaces between synaptic regions decreased in size at the same time and to a similar extent as the regions themselves. Following testosterone replacement, the spaces between synaptic regions expanded and each existing ACh receptor region enlarged. Ultrastructural analysis showed that there was no loss or addition of postsynaptic secondary junctional folds in the muscle fiber membrane (where ACh receptors are located) as junctions shrank and expanded. Rather, folds became more densely packed as muscle fibers atrophied following castration and less densely packed as muscle fibers hypertrophied following testosterone replacement. From these studies of the bulbocavernosus muscle, as from our previous studies of the sternomastoid muscle, we conclude that neuromuscular junction size is directly coupled to muscle fiber size. Androgens modulate muscle fiber volume directly, leading to a change in the surface area of the muscle fiber membrane, which in turn causes the postsynaptic specializations to shrink or expand. The concomitant shrinkage and stretching of motor nerve terminals that we observed can only be accounted for by their adhesion to postsynaptic specializations that are also changing size. Thus adhesion, rather than an interchange of diffusible factors, trophic or otherwise, is likely to be the primary determinant of coordinated pre- and postsynaptic enlargement in growing mammalian skeletal muscles.

Androgens↗
PubMed1988

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)

Animals↗
PubMed1988

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.

Animals↗
PubMed1987

Selective innervation of types of fibres in developing rat muscle.

The technique of glycogen depletion has been used to identify the types of muscle fibres innervated by individual motor neurones in the neonatal rat. This analysis shows that neonatal motor units are highly biased in their fibre type composition, even at times when the fibres receive extensive polyneuronal innervation. This finding suggests that the innervation of muscle fibres is somehow sorted according to type during early development. This sorting does not appear to occur during the removal of the polyneuronal innervation because little, if any, increase in the bias of unit compositions occurs as the number of synapses present in the muscle is reduced 2- to 3-fold. To determine whether the sorted innervation might be explained by a selective synaptogenesis, a study was made of the type compositions of units formed by reinnervation of neonatal soleus muscle. Glycogen depletion of single units 2 weeks following crush of the soleus nerve at postnatal day 2 showed that most of them (10/12) had biased type compositions which could not be explained by a random reinnervation. The location of fibres in the reinnervated motor units suggests that the regenerating axons innervated a novel set of fibres. The differentiation of fibres into types was apparently not changed during their reinnervation. These results imply that regenerating motor neurones in the neonatal rat selectively reinnervate muscle fibre types. These and other studies further imply that the organization of fibres into motor units during normal development does not occur, as is widely believed, by a random innervation of naive fibres and their subsequent differentiation under the influence of innervation.

Animals↗