Muscle spindles constantly report skeletal muscle length and movement. Mechanosensory transduction occurs at annulospiral terminals around intrafusal muscle fibres. We reported that Ca2+-dependent glutamate release from synaptic-like vesicles in these terminals modulates spindle firing (Bewick et al., 2005). To test if voltage-gated Ca2+ channels (VGCCs) or Ca2+-dependent K+ channels (KCa) are also functionally important, we tested whether channel-selective neurotoxins affected spindle afferent discharge. Adult Sprague-Dawley rats (male, 300-370 g) were killed (Schedule 1, Animal (Scientific Procedures) Act, 1986) and 4th lumbrical nerve-muscle preparations excised and placed in gassed (95%O2-5%CO2) Liley`s saline. Spindle afferent discharges were recorded en passant with Ag wire electrodes and firing frequency (mean ± SE, n) determined for the first 0.5 s of the “hold” phase of stretch-and-hold cycles (~10% muscle length) for ‘n’ preparations. The significance of differences between pre-drug and with-drug means was evaluated by paired t-test, with a threshold of P < 0.05. Unlike inorganic Ca2+ channel blockers Co2+ and Ni2+/Cd2+, which abolish responses (Bewick et al., 2005), P/Q type VGCC inhibitors enhanced firing. ¿-Agatoxin-IVA (200 nM, P/Q type) increased firing to 301% of control (92.7 ± 15.8 imp/s vs 278.6 ± 23.9 imp/s, 6; P < 0.0001). ¿-Conotoxin-MVIIC (1 µM, Q type) enhanced firing to 202% (98.40 ± 21.83 imp/s vs 199.60 ± 24.62 imp/s, 5; P < 0.002). Conversely, ¿-conotoxin-GVIA (1 µM; N type) had no significant effect (176.92 ± 14.77 imp/s vs 196.92 ± 10.47 imp/s, 6; P = 0.1). L-type blockers Taicatoxin (50 nM, 4) and Nifedipine (10 µM, 5) increased afferent firing (131.88 ± 10.05 imp/s vs 302.13 ± 25.17 imp/s and 192.00 ± 13.09 imp/s vs 271.70 ± 9.56 imp/s, respectively) but also caused spontaneous muscle twitching, suggesting actions on skeletal muscle dihydropyridine receptors. Thus, only P/Q type channel blockers increased afferent discharge in the absence of other effects. P/Q type channels often regulate KCa (BK or SK) channel opening (Edgerton & Reinhart, 2003). Therefore, selective KCa channel blockers were applied. Charybdotoxin (200 nM; SK & BK), iberiotoxin (200 nM; BK selective) and apamin (200 nM; SK selective) all increased afferent discharge (to 195%, 4; 224%, 4 & 160%, 6 of control, respectively; all P < 0.02). Conversely, NS1619 (BK channel activator) inhibited firing (1 µM, 179.75 ± 14.35 imp/s vs 33.25 ± 18.44 imp/s, 6). This was not due to voltage-gated potassium channel or action potential conduction block, since NS1619 did not affect Nervus saphenus compound action potential amplitude (100 µM, 4). The data suggest Ca2+ entry through P/Q type channels activates KCa channels to regulate firing frequency in spindle 1a afferents.
|Journal||Proceedings of the Physiological Society|
|Publication status||Published - 2009|