Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCB248

Poster Communications

Transient receptor potential channel V4 ligands differentially modulate dynamic and static responses of stretch-evoked firing in ex vivo rat muscle spindles

M. Gniss1,2, R. A. Lofthouse1, P. Miti1, F. C. Shenton3, R. W. Banks3, G. S. Bewick1

1. School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Aberdeen, United Kingdom. 2. Clinical Neuroscience, University of Cambridge, Cambridge, United Kingdom. 3. School of Biological & Biomedical Sciences, Durham University, Durham, United Kingdom.

Mammalian muscle spindle primary sensory terminals are exquisitely sensitive to both static length and change of length. How the nerve terminals transduce these mechanical stimuli is still unclear. While piezo2 knock out (1) clearly profoundly affects spindle stretch-evoked firing, the sensory terminals contain a large range of other ion channels whose functions need to be explored. The vanilloid-type TRPs (TRPVs) are of particular interest to mechansensation (2), particularly TRPV4, which is expressed in hair cells of rodents (3) and zebrafish (4). We have therefore examined a putative mechanosensory role for TRPV4 in rat muscle spindles. Fourth deep lumbrical nerve-muscle preparations were dissected from adult male Sprague-Dawley rats (246g-389g) killed by anaesthetic overdose (150-200mg/kg phenopentobarbital, i.p.), then kept in artificial cerebrospinal fluid saturated with 95% O2/5% CO2 at room temperature. Stretch-evoked afferent discharges were recorded during trapezoidal (1 sec ramp - 3 sec hold - 1 sec release) and saw-tooth (25 @ 0.2Hz, 50 @ 1Hz & 50 @ 5Hz) 1mm changes in muscle length (~10% total length) from an electromechanical puller. Total action potential (AP) counts were made during movement (dynamic) and hold (static) phases in drug-free and rising concentrations of ligands (1hr in each). Data were analysed by 1-way repeated measures ANOVA, with Holm-Sidak-corrected post-test comparison between means and a significance threshold of P<0.05 for ‘n' muscles. The broad spectrum TRP channel blocker 2-aminoethyl borate abolished static-phase firing (121.9±28.2 AP/s pre-drug) at 100 µM (P<0.001, n=3), but increasing rates of stretch (dynamic firing) were progressively more resistant to inhibition (0.2Hz: pre-drug 341.7±51.2 vs 100 µM 0.7 ± 0.3 APs/cycle; P<0.001. 5Hz: pre-drug 13.7±3.1 vs 100 µM 3.7±2.1 APs/cycle; P<0.05). Lower concentrations (1 & 10 µM) had no significant effect on any firing counts. Ligands selective for TRPV4 were then tested. RN-1734 (TRPV4 antagonist, n=4) inhibited static-phase firing at 3µM (pre-drug 134.9±7.1 APs/sec vs 89.1±6.0 APs/sec; P<0.05), but 10µM was needed to inhibit dynamic ramp, 0.2Hz and 1Hz responses (each P<0.05), while 30µM was needed to reduce 5Hz-evoked firing (16.2±1.9 vs 5.7±2.2 APs/cycle; P<0.05). Interestingly, the potent TRPV4 agonist GSK1016790A did not affect firing up to 200nM (n=7), but it blocked 10µM RN-1734's inhibition of trapezoid-induced dynamic firing (P=0.44, n=4). Immunofluorescence provided further support for TRPV4 expression on spindle primary sensory nerve terminals. These data suggest the putative mechanotransduction channel TRPV4 is present on spindle sensory nerve terminals, where blockade preferentially inhibits static and slow dynamic firing.

Where applicable, experiments conform with Society ethical requirements