The identity and properties of the mechanotransduction channel within muscle spindles, the proprioceptive sensory organs reporting skeletal muscle length, are poorly understood. We have shown previously that stretch releases glutamate from synaptic-like vesicles (SLVs) within the spindle terminals, activating an unusual metabotropic glutamate receptor (mGluR) linked to phospholipase D and increasing afferent firing (Bewick et al, 2005). However, the stretch activated channel(s) causing the initial depolarization and triggering SLV fusion with the membrane remains unidentified. To further characterise these channels we tested the effects of gadolinium (Gd3+), a lanthanoid, and FM1-43, a styryl pyridinium dye, which both block electrical currents mediated through stretch-activated channels in other systems (Yang & Sachs, 1989; Hajduczok et al, 1994; Gale et al, 2001; Drew & Wood, 2007).Adult male Sprague-Dawley rats (360-430 gm) were humanely killed then 4th lumbrical nerve-muscle preparations excised and equilibrated under gassed (95%O2-5%CO2) saline. Spindle discharges were recorded en passant from the muscle nerve with Ag+ wire electrodes at room temperature during 1 mm stretch-and-hold cycles (~10% muscle length). The effect of increasing concentrations of Gd3+ and FM1-43 applied for at least 60 min on stretch-evoked firing was compared to responses in saline alone.Differences in mean firing frequencies (impulses per second, imp/sec) between the drug-free and in-drug conditions were evaluated by paired t-test or one way ANOVA followed by Tukey multiple comparison post-tests, as appropriate, with a significance threshold of P = 0.05.100 µM Gd3+ decreased firing from 353.4 ± 23.6 to 272.3 ± 16.2 imp/sec (mean ± SE; n = 6, P < 0.01) after 1 hr incubation. 1 mM Gd3+ did not decrease the firing rate further but 10 mM abolished firing altogether (P < 0.0001). Some firing returned after 1 hr wash. However, the firing rates (29.4 ± 12.0 imp/sec) were well below pre-treatment rates. In contrast, 5 μM FM1-43 had no significant effect on firing (from 267.1 ± 6.8 to 286.9 ± 12.5 imp/sec, n = 3; P = 0.41 at 3 hr). At 10 μM, the dye decreased firing by ~30% (to 152.8 ± 39.5 imp/sec; P < 0.01) after 3 hrs, a concentration producing ~80% block of currents in cultured sensory neurones (Drew et al., 2007). Dye does penetrate the selectively permeable spindle capsule under these conditions, as 5 μM FM1-43 labels these terminals in only 2 hr (Bewick et al, 2005). These data indicate that stretch-activated channels that initiate stretch-activated afferent discharges in muscle spindles are Gd3+ sensitive but relatively insensitive to FM1-43.
Watson, S., Aryiku, C-L., Banks, R. W., & Bewick, G. S. (2010). Comparison of gadolinium and FM1-43 as blockers of stretch-evoked firing of rat muscle spindle afferents. Proceedings of the Physiological Society, 21, [PC21].