Comparison of gadolinium and FM1-43 as blockers of stretch-evoked firing of rat muscle spindle afferents

Sonia Watson, Cheryl-Lee Aryiku, Robert W Banks, Guy Smith Bewick

Research output: Contribution to journalAbstract

Abstract

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.
Original languageEnglish
Article numberPC21
JournalProceedings of the Physiological Society
Volume21
Publication statusPublished - 2010
EventPhysiological Society Cross-Themed Meeting - Durham, United Kingdom
Duration: 15 Dec 201017 Dec 2010

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Muscle Spindles
Gadolinium
Coloring Agents
Synaptic Vesicles
Muscles
Phospholipase D
Lanthanoid Series Elements
Metabotropic Glutamate Receptors
Membrane Fusion
Neuromuscular Junction
Sensory Receptor Cells
Pharmaceutical Preparations
Capsules
Sprague Dawley Rats
Glutamic Acid
Analysis of Variance
Electrodes
Skeletal Muscle
FM1 43
Temperature

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Comparison of gadolinium and FM1-43 as blockers of stretch-evoked firing of rat muscle spindle afferents. / Watson, Sonia; Aryiku, Cheryl-Lee; Banks, Robert W; Bewick, Guy Smith.

In: Proceedings of the Physiological Society, Vol. 21, PC21, 2010.

Research output: Contribution to journalAbstract

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title = "Comparison of gadolinium and FM1-43 as blockers of stretch-evoked firing of rat muscle spindle afferents",
abstract = "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.",
author = "Sonia Watson and Cheryl-Lee Aryiku and Banks, {Robert W} and Bewick, {Guy Smith}",
year = "2010",
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T1 - Comparison of gadolinium and FM1-43 as blockers of stretch-evoked firing of rat muscle spindle afferents

AU - Watson, Sonia

AU - Aryiku, Cheryl-Lee

AU - Banks, Robert W

AU - Bewick, Guy Smith

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

M3 - Abstract

VL - 21

JO - Proceedings of the Physiological Society

JF - Proceedings of the Physiological Society

SN - 1749-6187

M1 - PC21

ER -