Intra-neuronal influences on development of the mammalian neuromuscular junction

During development of the nervous system an excess number of synapses are formed,most of which are subsequently pruned, resulting in functional neural networks. Theprecise mechanisms that determine which synapses are formed and which synapses aremaintained are not thoroughly understood. The aim of this thesis is to investigate theintra-neuronal constraints and influences on synapse formation and elimination duringdevelopment.In the first part of the thesis I investigated intra-neuronal influences on synapse elim-ination. Synapse elimination is known to occur at polyneuronally innervated neuro-muscular junctions through competition, leading to mononeuronally innervated mus-cle fibres. However, whether synapse elimination ever occurs in the absence of com-petition, leading to muscle fibres becoming denervated, has not been resolved. Thedata presented in this thesis suggest that only large motor units undergo a reductionin motor unit size in the absence of competition. Using the Rasmussen and Willshaw(1993) version of the Dual Constraint Model I show that these data are consistent withthe prediction that synapses will be eliminated from muscle fibres when a neuron’s re-sources become stretched, for instance as a result of the normal growth of the animal.Larger motor units, which innervate more synapses, will thus be more vulnerable tothe extra demand put upon them by the growth of each synapse. The model predictsthat synapse elimination in the absence of competition should occur at least over thefirst 6 months of life and not only during the first two postnatal weeks, when mostpolyneuronal innervation is normally eliminated.In the second part of the thesis, I investigated intra-neuronal influences on synapseformation. Specifically I tested the hypothesis that each branch of a motor neuronforms synapses randomly and independently of other branches. If true there shouldbe instances where two branches from the same neuron initially innervate the sameendplate (sibling branch convergence). Sibling branch convergence was experimen-tally investigated in both regenerating and developing motor neurons. The evidencesuggests that sibling branches can converge on the same muscle fibre and that theycan competitively eliminate each other. However, it appears that convergence does notoccur at the frequency that would be expected, suggesting that branches from the sameiii motor neuron do not form synapses independently of each other.At present there are limitations in imaging immature networks due to the spatial resolu-tion limit of light microscopy. The last part of this thesis explores thin serial sectioningand reconstruction as a possible technique for increasing resolution in the z-axis. Thistechnique has potential to be developed but I show that at present it does not providesufficient resolution to discriminate between developing motor axons in neonatal lum-brical muscles

Analysis of local and global topographic order in mouse retinocollicular maps

We introduce the Lattice Method for the quantitative assessment of the topographic order within the pattern of connections between two structures. We apply this method to published visuocollicular mapping data obtained by Fourier-based intrinsic imaging of mouse colliculus. We find that, in maps from wild types and β2 knock-outs, at least 150 points on the colliculus are represented in the visual field in the correct relative order. In maps from animals with knock-out of the three ephrinA ligands (TKO), thought to specify the rostrocaudal axis of the map, the projection on the colliculus of each small circular area of visual field is elongated approximately rostrocaudally. Of these projections, 9% are made up of two distinct regions lying along the direction of ingrowth of retinal fibers. These are similar to the ectopic projections found in other ephrinA knock-out data. Coexisting with the ectopic projections, each TKO map contains a submap where neighbor–neighbor relations are preserved, which is ordered along both rostrocaudal and mediolateral axes, in the orientation found in wild-type maps. The submaps vary in size with order well above chance level, which can approach the order in wild-type maps. Knock-out of both β2 and two of the three ephrinAs yields maps with some order. The ordered TKO maps cannot be produced by correlated neural activity acting alone, as this mechanism is unable to specify map orientation. These results invite reassessment of the role of molecular signaling, particularly that of ephrinAs, in the formation of ordered nerve connections

Segmentation of the mouse fourth deep lumbrical muscle connectome reveals concentric organization of motor units

Connectomic analysis of the nervous system aims to discover and establish principles that underpin normal and abnormal neural connectivity and function. Here we performed image analysis of motor unit connectivity in the fourth deep lumbrical muscle (4DL) of mice, using transgenic expression of fluorescent protein in motor neurones as a morphological reporter. We developed a method that accelerated segmentation of confocal image projections of 4DL motor units, by applying high resolution (63×, 1.4 NA objective) imaging or deconvolution only where either proved necessary, in order to resolve axon crossings that produced ambiguities in the correct assignment of axon terminals to identified motor units imaged at lower optical resolution (40×, 1.3 NA). The 4DL muscles contained between 4 and 9 motor units and motor unit sizes ranged in distribution from 3 to 111 motor nerve terminals per unit. Several structural properties of the motor units were consistent with those reported in other muscles, including suboptimal wiring length and distribution of motor unit size. Surprisingly, however, small motor units were confined to a region of the muscle near the nerve entry point, whereas their larger counterparts were progressively more widely dispersed, suggesting a previously unrecognised form of segregated motor innervation in this muscle. We also found small but significant differences in variance of motor endplate length in motor units, which correlated weakly with their motor unit size. Thus, our connectomic analysis has revealed a pattern of concentric innervation that may perhaps also exist in other, cylindrical muscles that have not previously been thought to show segregated motor unit organisation. This organisation may be the outcome of competition during postnatal development based on intrinsic neuronal differences in synaptic size or synaptic strength that generates a territorial hierarchy in motor unit size and disposition

Activity-dependent degeneration of axotomized neuromuscular synapses in Wld(S) mice

AbstractActivity and disuse of synapses are thought to influence progression of several neurodegenerative diseases in which synaptic degeneration is an early sign. Here we tested whether stimulation or disuse renders neuromuscular synapses more or less vulnerable to degeneration, using axotomy as a robust trigger. We took advantage of the slow synaptic degeneration phenotype of axotomized neuromuscular junctions in flexor digitorum brevis (FDB) and deep lumbrical (DL) muscles of Wallerian degeneration-Slow (WldS) mutant mice. First, we maintained ex vivo FDB and DL nerve-muscle explants at 32°C for up to 48h. About 90% of fibers from WldS mice remained innervated, compared with about 36% in wild-type muscles at the 24-h checkpoint. Periodic high-frequency nerve stimulation (100Hz: 1s/100s) reduced synaptic protection in WldS preparations by about 50%. This effect was abolished in reduced Ca2+ solutions. Next, we assayed FDB and DL innervation after 7days of complete tetrodotoxin (TTX)-block of sciatic nerve conduction in vivo, followed by tibial nerve axotomy. Five days later, only about 9% of motor endplates remained innervated in the paralyzed muscles, compared with about 50% in 5day-axotomized muscles from saline-control-treated WldS mice with no conditioning nerve block. Finally, we gave mice access to running wheels for up to 4weeks prior to axotomy. Surprisingly, exercising WldS mice ad libitum for 4weeks increased about twofold the amount of subsequent axotomy-induced synaptic degeneration. Together, the data suggest that vulnerability of mature neuromuscular synapses to axotomy, a potent neurodegenerative trigger, may be enhanced bimodally, either by disuse or by hyperactivity