Intra-neuronal influences on development of the mammalian neuromuscular junction

Abstract

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.