Transforming growth factor-βs (TGF-βs) are highly expressed in neural development but why the adult nervous system continues to express them is unclear. TGF-β2 is concentrated at mature neuromuscular junctions (NMJs) of mammalian skeletal muscle fibres, and the nerve terminal expresses TβR-II receptors. To test the role of TGF-β2 at mammalian NMJs, I performed four experiments. The first study tested whether TGF-β2 acutely modulates synaptic transmission at mature mammalian NMJs. Second, I asked if chronically reduced TGF-β2 expression disrupts synaptic transmission. Third, I asked if TGF-β2’s effects differ in terminals adapted to different activity patterns in vivo. Lastly, I asked whether TGF-β1, a related peptide to TGF-β2, is distinct in terms of its effects on transmitter release. Using single electrode potential recording, I found TGF-β2 significantly increased the amplitude of spontaneous released single neurotransmitter vesicles (miniature endplate potentials, MEPPs) and nerve stimulation evoked multi-vesicular release (endplate potentials, EPPs). These effects were blocked by L-vesamicol, a vesicular acetylcholine transporter inhibitor, and bafilomycin, a proton pump inhibitor, suggesting the increase in MEPP/EPP amplitude is due to increased vesicle filling presynaptically. These effects were also blocked by the MARK inhibitors, UO126 and PD98059, suggesting TGF-β2 acts via a MARK-dependent pathway. Postsynaptically, two electrode recording showed postsynaptic potential amplitude was enhanced by an increased fibre input resistance, suggesting TGF-β2 also acts postsynaptically. TGF-β2 reduced the number of vesicles released per stimulus (quanta content, QC) but this was blocked by atropine, showing this was indirect through autoreceptor negative feedback. Voltage clamp recording showed TGF-β2 significantly increased the miniature end plate currents (MEPCs), but not the end-plate currents (EPCs), supporting my initial hypothesis that TGF-β2 acts mainly presynaptically to increase vesicle filling. In TGF-β2+/- mice, I found greater MEPP amplitude variability. This supports my previous findings that TGF-β2 modulates vesicle filling. Unexpectedly, there was an excess in larger MEPP sizes (>0.88 mV), perhaps reflecting upregulation of either presynaptic signalling or another synaptic mediator. Two MEPP amplitude populations were induced in TGF-β2-treated TGF-β2+/- mouse NMJs, similar to the bimodal vesicle population in electroplaques. The extensor digitorum longus (EDL, ~95% fast fibres) and soleus (SOL, ~95% slow fibres) were used to investigate whether the TGF-β2-mediated effect differed between fibre types. Overall, TGF-β2 increased the quantal size (MEPP amplitude) in NMJs of both muscles, suggesting this effect is not fibre-type specific and, together with results in mice, that the TGF-β2-mediated increase in vesicle filling is common to all mammalian neuromuscular terminals. With respect to EPP amplitude and QC, the results differed between muscles. In EDL, the EPP amplitude was not significantly changed, whereas it increased in SOL. In EDL, QC was reduced but not in SOL. These difference compared to diaphragm perhaps do reflect muscle fibre-type dependent differences. TGF-β1, at 0.1 ng/ml, significantly reduced quantal size – the opposite of TGF-β2 at any concentration. One explanation would be that a receptor inhibition by TGF-β1 at low concentration interferes with endogenous TGF-β2 binding/receptor activation at the NMJ. However, when the TGF-β1 concentration was raised to 1 ng/ml, like TGF-β2, it significantly increased MEPP amplitude. This suggests that perhaps sufficient binding of TGF-β1 results in the receptor activation of TGF-β2 like signalling. Overall, I conclude that TGF-β2 enhances the size of spontaneous synaptic potentials in all types of muscle fibres, and this is much more rapid (1 hr vs 1 day) than at central neurone synapses in culture. Detailed study in the rat diaphragm shows it increased the evoked EPP amplitude, reduced QC and increased postsynaptic input resistance. Together, TGF-β2 would therefore enhance the postsynaptic depolarisation increasing synaptic strength, and by reducing QC, increase the efficiency of neurotransmission at mammalian NMJs. While unimportant for single stimuli in healthy terminals, by conserving vesicle use, it may help maintain release during stimulus trains, especially during neuromuscular disease.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:521182 |
| Date | January 2009 |
| Creators | Fong, Sitt Wai |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=133996 |
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