Proceedings of The Physiological Society

University of Leeds (2008) Proc Physiol Soc 10, PC4

Poster Communications

Transforming growth factor-β2 increases synaptic vesicle loading at rat neuromuscular junctions

S. Fong1, I. S. McLennan2, G. S. Bewick1

1. School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom. 2. Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand.

The transforming growth factor-β (TGF-β) cytokine family has three mammalian isoforms; TGF-βs 1-3. They play important roles in neuromuscular development (Böttner et al., 2000) and axonal outgrowth (McLennan et al., 1994) in embryos. However, they are also expressed in adults. Recently, TGF-β1 and -β2 have been shown to enhance transmission between neurones in culture (Fukushima et al., 2007). TGF-β2 is highly expressed at neuromuscular junctions (NMJs) of mammalian skeletal muscle fibres (McLennan & Koishi, 2002), while the overlying motor nerve terminal expresse the receptor TβR-II (Jiang et al., 2000). We therefore asked whether TGF-β2 modulates synaptic transmission at mature mammalian NMJs. Adult male Lister Hooded rats (230-350gm) were stunned then killed by cervical dislocation, in accordance with Schedule 1 of the UK Animals (Scientific Procedures) Act, 1986. Rat hemidiaphragm-nerve preparations were dissected and the muscle fibres paralysed with 2 µM µ-conotoxin GIIIB. The amplitude of spontaneous miniature and evoked postsynaptic potentials (MEPPs and EPPs, respectively) were recorded by standard intracellular sharp electrode electrophysiology, from 1 hr after applying BSA (control) or TGF-β2 (1 ng/ml). The quantal content (QC, vesicles per EPP) was calculated by the direct method, with non-linear summation correction. Data are shown as mean ± SEM, and number of muscle fibres, using 3-6 rats per group. Differences between means were compared using a two sample Student’s t-test. P < 0.05 was taken as significant. Compared to BSA, TGF-β2 increased the amplitude of both MEPPs (0.44 mV ± 0.03, n = 33 and 0.64 mV ± 0.03, n = 46, P < 0.0001) and EPPs (21.6 mV ± 1.2, n = 33 and 25.6 mV ± 0.9, n = 37, P < 0.001). In contrast, it decreased the mean QC (43.1 mV ± 1.9, n = 33 and 32.9 mV ± 1.7, n= 37, P < 0.0001). TGF-β2 caused no significant change in EPP time course (time to return to 50 % peak amplitude, 2.88 ms ± 0.08, n = 33 and 3.0 ms ± 0.08, n = 37, P = 0.3) or resting membrane potential (-65.0 mV ± 0.7, n = 47 and -64.8 mV ± 0.5, n = 46, P = 0.8,). However, the effects on transmission were all blocked by the vesicular acetylcholine transport inhibitors, L-vesamicol (5 μM; MEPP, 0.43 mV ± 0.03, n = 21, P = 0.8; EPP, 20.7 mV ± 2.1, P = 0.7, n = 21; QC, 39.3 mV ± 2.3, P = 0.2, n = 21) and bafilomycin (0.1 μM). Thus, these effects of TGF-β2 seem due primarily to increased vesicular transmitter loading. Overall, the strength, security and efficiency of neurotransmission at mature NMJs were increased. TGF-β2 produces synaptic effects much more rapidly at mature mammalian NMJs (1-2 hrs) compared CNS neuronal cultures (1-2 days). This rapid action in the absence of motor neuronal cell bodies suggests a local signalling mechanism without alterations in neuronal gene expression.

Where applicable, experiments conform with Society ethical requirements