Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death (PCD). How motoneurons are selected to live or die during this developmental period is unknown. Motoneuron survival depends on the presence of both their target skeletal muscle and the Schwann cells that surround the peripheral axons. These target bskeletal muscles and Schwann cells contain trophic factors that are essential for the survival of motoneurons. Therefore it is postulated that motoneuron survival depends on its ability to access these trophic factors.
Motoneurons may utilize the neuromuscular synapse to compete for a limited supply of muscle-derived trophic factors. Transplanting embryonic chick limbs in place of quail limbs and vice versa changes motoneuron survival in direct proportion with the number of neuromuscular synapses. Similarly, motoneuron survival is increased in proportion to increased intramuscular axonal branching and neuromuscular synapse number as a consequence of decreased skeletal muscle activity. Furthermore, the onset of motoneuron death is coincident with the formation of neuromuscular synapses. Thus, the neuromuscular synapse may influence the fate of motoneurons during embryonic development.
The aim of my PhD thesis is to test how neuromuscular synapse formation affects motoneuron survival during embryonic development. Neuromuscular synapses are highly specialized chemical synapses where fast and reliable transmission is provided by the direct apposition of synaptic vesicle release sites in the presynaptic nerve terminal that lie directly adjacent to acetylcholine receptor (AChR) clusters in the postsynaptic membrane. These synaptic specializations are initiated and maintained by the heparin sulfate proteoglycan, agrin. Agrin acts through its muscle specific kinase (MuSK) receptor to initiate AChR clustering through the receptor associated protein of the synapse (rapsyn). The agrin-induced postsynaptic apparatus is thought to provide for an as yet unknown retrograde signal that subsequently initiates presynaptic differentiation. Genetically modified mice that lack agrin have few extrasynaptic AChR clusters whereas mice that lack MuSK or rapsyn have no AChR clusters. All motor axons in these mutant mice branch more and extend further than in wild-type mice. In addition, agrin and MuSK promote increased gene transcription from subsynaptic muscle nuclei in wildtype mice. Accordingly, agrin and MuSK-deficient mice do not show preferential AChR transcription in the centre of muscle and have AChRs distributed diffusely throughout the muscle. Rapsyn however, retains gene transcription in centrally located muscle nuclei and thus retains a high concentration of non-clustered AChRs in the centre of muscle. These neuromuscular defects ensure the mutant mice die near birth resulting from their inability to breath. In this thesis I quantify and compare motoneuron survival, motor axon branching and skeletal muscle contraction in agrin and rapsyn-deficient mice.
There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared to their wild-type littermates at embryonic day 18.5 (El8.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.
The second aim of this study was to evaluate the contribution of target-derived trophic factors to increases in motor axon branching and number, in rapsyn-deficient mice. We (Selena Bartlett and I) have used reverse transcription-polymerase chain reaction and Western blot to document the expression of known trophic factors and their receptors in muscle, during the period of synapse formation in rapsyn-deficient mouse embryos. We found that the mRNA levels for ciliary neurotrophic factor (CNTF) was decreased in the rapsyn-deficient muscles compared with littermate controls while those for NGF, BDNF, NT-3 and TGF-β2 did not differ. In addition both the mRNA and the protein expression for suppressor of cytokine signalling 3 (S0CS3) decreased while janus kinase 2 (JAK2) did not change in the rapsyn-deficient muscles compared with littermate controls. This suggests that disruption of the development of the postsynaptic specialization resulted in alteration of the CNTF signaling pathway within the target for motoneurons, skeletal muscle. This alteration may in part, account for the increased intramuscular axonal branching and motoneuron survival seen in rapsyn-deficient mice.
The third and final aim of this study was to test whether pre-motor circuits regulate the number of surviving motoneurons by controlling motor unit activity. To achieve this, I counted the number of motoneurons in gephyrin-deficient mice. Gephyrin-deficient mice lack glycine and most GABAA receptor clusters throughout the CNS. Motoneuron survival was significantly increased in the spinal cord in the brachial (22%) and lumbar (37%) lateral motor columns. In contrast, motoneuron survival was decreased in respiratory motor nuclei including the hypoglossal (21%), trigeminal (15%) and phrenic (30%) motor nuclei. The decrease in phrenic motoneuron survival was accompanied with decreases in innervation of diaphragm skeletal muscle. These changes in motoneuron survival and innervation are likely to be a result of changed motor unit activity that is brought about by the loss of glycine and GABAA receptor clusters. The regional differences in motoneuron survival in gephyrin-deficient mice compared to wild-type mice suggest that GABA and glycine regulate excitatory neurotransmission in spinal motor circuits and inhibitory neurotransmission in pre-motor respiratory networks in vivo during the period of PCD.