A role for glutamate in regulating mechanosensory sensitivity

Further evidence from rat muscle spindle primary afferent terminals

Anna Simon, Robert W Banks, Guy Smith Bewick

Research output: Contribution to journalAbstract

Abstract

Muscle spindles are proprioceptive sensory organs that constantly report the length and movements of skeletal muscle. The complicated process of mechanotransduction underlying their function is still poorly understood. The stretch-sensitive annulospiral terminals have populations of small (50 nm), clear synaptic-like vesicles (SLVs). We have shown (Bewick et al., 2005) SLVs contain glutamate, which when released, increases spindle firing by acting on a non-canonical, phospholipase D-coupled metabotropic glutamate receptor (PLD-mGluR). For a better understanding of how glutamate modulates afferent firing, we examined the effects of DL-TBOA, a non-selective excitatory amino acid transporter (EAAT) inhibitor and Rose Bengal, a blocker of the vesicular glutamate transporters (VGLUTs). Adult Sprague-Dawley rats (male, 300-370g) were killed and 4th lumbrical nerve-muscle preparations excised from both hind legs and stored under gassed (95%O2-5%CO2) saline. Spindle discharges were recorded at room temperature during 1 mm stretch-and-hold cycles (~10% muscle length). Data are expressed as mean ± SD. Differences between the pre-drug control and with-drug mean firing frequencies (impulses per second, imp/sec) were evaluated by paired t-test, with a significance threshold of P = 0.05. 100 µM DL-TBOA increased afferent firing from 199.0 ± 48.3 to 322.9 ± 48.9 imp/sec (n = 8; P < 0.0001) after 3 hr incubation, indicating that EAATs are important regulators of the effects of endogenously-released glutamate. This excitation involved activation of PLD-mGluRs, since it could be counteracted by PCCG-13, a specific PLD-mGluR blocker. Thus, when lumbricals were incubated in DL-TBOA with 10 µM PCCG-13, firing initially increased, from 214.6 ± 19.4 to 273.6 ± 27.7 imp/sec (2 hr, n = 2; P < 0.002), but then decreased to 123.5 ± 28.6 imp/sec (3 hr, n = 2; P < 0.003). All effects were fully reversible on washing. However, the non-selective VGLUT inhibitor Rose Bengal had no significant effect on the afferent discharge at 100 nM (n = 2; P < 0.51) or 1 µM (n = 2; P < 0.11). These data indicate tonic endogenous glutamate release from SLV recycling constantly modulates firing in muscle spindle afferents, with EAATs limiting its effects by re-uptake. The absence of perturbation from the VGLUT inhibitor, despite the presence of VGLUT1 (Wu et al., 2004), requires further study. It may indicate a large pool of pre-loaded SLVs must first be emptied before glutamate depletion effects are observed.
Original languageEnglish
Article numberC120
JournalProceedings of the Physiological Society
Volume19
Publication statusPublished - 2010

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Muscle Spindles
Vesicular Glutamate Transport Proteins
Synaptic Vesicles
Glutamic Acid
Rose Bengal
Phospholipase D
Metabotropic Glutamate Receptors
Amino Acid Transport Systems
Excitatory Amino Acids
Neuromuscular Junction
Drug and Narcotic Control
Sprague Dawley Rats
Leg
Skeletal Muscle
Muscles
Temperature
Pharmaceutical Preparations
Population
benzyloxyaspartate

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@article{0cfcb5fbb2734bd3b9de25be8a54b1e9,
title = "A role for glutamate in regulating mechanosensory sensitivity: Further evidence from rat muscle spindle primary afferent terminals",
abstract = "Muscle spindles are proprioceptive sensory organs that constantly report the length and movements of skeletal muscle. The complicated process of mechanotransduction underlying their function is still poorly understood. The stretch-sensitive annulospiral terminals have populations of small (50 nm), clear synaptic-like vesicles (SLVs). We have shown (Bewick et al., 2005) SLVs contain glutamate, which when released, increases spindle firing by acting on a non-canonical, phospholipase D-coupled metabotropic glutamate receptor (PLD-mGluR). For a better understanding of how glutamate modulates afferent firing, we examined the effects of DL-TBOA, a non-selective excitatory amino acid transporter (EAAT) inhibitor and Rose Bengal, a blocker of the vesicular glutamate transporters (VGLUTs). Adult Sprague-Dawley rats (male, 300-370g) were killed and 4th lumbrical nerve-muscle preparations excised from both hind legs and stored under gassed (95{\%}O2-5{\%}CO2) saline. Spindle discharges were recorded at room temperature during 1 mm stretch-and-hold cycles (~10{\%} muscle length). Data are expressed as mean ± SD. Differences between the pre-drug control and with-drug mean firing frequencies (impulses per second, imp/sec) were evaluated by paired t-test, with a significance threshold of P = 0.05. 100 µM DL-TBOA increased afferent firing from 199.0 ± 48.3 to 322.9 ± 48.9 imp/sec (n = 8; P < 0.0001) after 3 hr incubation, indicating that EAATs are important regulators of the effects of endogenously-released glutamate. This excitation involved activation of PLD-mGluRs, since it could be counteracted by PCCG-13, a specific PLD-mGluR blocker. Thus, when lumbricals were incubated in DL-TBOA with 10 µM PCCG-13, firing initially increased, from 214.6 ± 19.4 to 273.6 ± 27.7 imp/sec (2 hr, n = 2; P < 0.002), but then decreased to 123.5 ± 28.6 imp/sec (3 hr, n = 2; P < 0.003). All effects were fully reversible on washing. However, the non-selective VGLUT inhibitor Rose Bengal had no significant effect on the afferent discharge at 100 nM (n = 2; P < 0.51) or 1 µM (n = 2; P < 0.11). These data indicate tonic endogenous glutamate release from SLV recycling constantly modulates firing in muscle spindle afferents, with EAATs limiting its effects by re-uptake. The absence of perturbation from the VGLUT inhibitor, despite the presence of VGLUT1 (Wu et al., 2004), requires further study. It may indicate a large pool of pre-loaded SLVs must first be emptied before glutamate depletion effects are observed.",
author = "Anna Simon and Banks, {Robert W} and Bewick, {Guy Smith}",
year = "2010",
language = "English",
volume = "19",
journal = "Proceedings of the Physiological Society",
issn = "1749-6187",

}

TY - JOUR

T1 - A role for glutamate in regulating mechanosensory sensitivity

T2 - Further evidence from rat muscle spindle primary afferent terminals

AU - Simon, Anna

AU - Banks, Robert W

AU - Bewick, Guy Smith

PY - 2010

Y1 - 2010

N2 - Muscle spindles are proprioceptive sensory organs that constantly report the length and movements of skeletal muscle. The complicated process of mechanotransduction underlying their function is still poorly understood. The stretch-sensitive annulospiral terminals have populations of small (50 nm), clear synaptic-like vesicles (SLVs). We have shown (Bewick et al., 2005) SLVs contain glutamate, which when released, increases spindle firing by acting on a non-canonical, phospholipase D-coupled metabotropic glutamate receptor (PLD-mGluR). For a better understanding of how glutamate modulates afferent firing, we examined the effects of DL-TBOA, a non-selective excitatory amino acid transporter (EAAT) inhibitor and Rose Bengal, a blocker of the vesicular glutamate transporters (VGLUTs). Adult Sprague-Dawley rats (male, 300-370g) were killed and 4th lumbrical nerve-muscle preparations excised from both hind legs and stored under gassed (95%O2-5%CO2) saline. Spindle discharges were recorded at room temperature during 1 mm stretch-and-hold cycles (~10% muscle length). Data are expressed as mean ± SD. Differences between the pre-drug control and with-drug mean firing frequencies (impulses per second, imp/sec) were evaluated by paired t-test, with a significance threshold of P = 0.05. 100 µM DL-TBOA increased afferent firing from 199.0 ± 48.3 to 322.9 ± 48.9 imp/sec (n = 8; P < 0.0001) after 3 hr incubation, indicating that EAATs are important regulators of the effects of endogenously-released glutamate. This excitation involved activation of PLD-mGluRs, since it could be counteracted by PCCG-13, a specific PLD-mGluR blocker. Thus, when lumbricals were incubated in DL-TBOA with 10 µM PCCG-13, firing initially increased, from 214.6 ± 19.4 to 273.6 ± 27.7 imp/sec (2 hr, n = 2; P < 0.002), but then decreased to 123.5 ± 28.6 imp/sec (3 hr, n = 2; P < 0.003). All effects were fully reversible on washing. However, the non-selective VGLUT inhibitor Rose Bengal had no significant effect on the afferent discharge at 100 nM (n = 2; P < 0.51) or 1 µM (n = 2; P < 0.11). These data indicate tonic endogenous glutamate release from SLV recycling constantly modulates firing in muscle spindle afferents, with EAATs limiting its effects by re-uptake. The absence of perturbation from the VGLUT inhibitor, despite the presence of VGLUT1 (Wu et al., 2004), requires further study. It may indicate a large pool of pre-loaded SLVs must first be emptied before glutamate depletion effects are observed.

AB - Muscle spindles are proprioceptive sensory organs that constantly report the length and movements of skeletal muscle. The complicated process of mechanotransduction underlying their function is still poorly understood. The stretch-sensitive annulospiral terminals have populations of small (50 nm), clear synaptic-like vesicles (SLVs). We have shown (Bewick et al., 2005) SLVs contain glutamate, which when released, increases spindle firing by acting on a non-canonical, phospholipase D-coupled metabotropic glutamate receptor (PLD-mGluR). For a better understanding of how glutamate modulates afferent firing, we examined the effects of DL-TBOA, a non-selective excitatory amino acid transporter (EAAT) inhibitor and Rose Bengal, a blocker of the vesicular glutamate transporters (VGLUTs). Adult Sprague-Dawley rats (male, 300-370g) were killed and 4th lumbrical nerve-muscle preparations excised from both hind legs and stored under gassed (95%O2-5%CO2) saline. Spindle discharges were recorded at room temperature during 1 mm stretch-and-hold cycles (~10% muscle length). Data are expressed as mean ± SD. Differences between the pre-drug control and with-drug mean firing frequencies (impulses per second, imp/sec) were evaluated by paired t-test, with a significance threshold of P = 0.05. 100 µM DL-TBOA increased afferent firing from 199.0 ± 48.3 to 322.9 ± 48.9 imp/sec (n = 8; P < 0.0001) after 3 hr incubation, indicating that EAATs are important regulators of the effects of endogenously-released glutamate. This excitation involved activation of PLD-mGluRs, since it could be counteracted by PCCG-13, a specific PLD-mGluR blocker. Thus, when lumbricals were incubated in DL-TBOA with 10 µM PCCG-13, firing initially increased, from 214.6 ± 19.4 to 273.6 ± 27.7 imp/sec (2 hr, n = 2; P < 0.002), but then decreased to 123.5 ± 28.6 imp/sec (3 hr, n = 2; P < 0.003). All effects were fully reversible on washing. However, the non-selective VGLUT inhibitor Rose Bengal had no significant effect on the afferent discharge at 100 nM (n = 2; P < 0.51) or 1 µM (n = 2; P < 0.11). These data indicate tonic endogenous glutamate release from SLV recycling constantly modulates firing in muscle spindle afferents, with EAATs limiting its effects by re-uptake. The absence of perturbation from the VGLUT inhibitor, despite the presence of VGLUT1 (Wu et al., 2004), requires further study. It may indicate a large pool of pre-loaded SLVs must first be emptied before glutamate depletion effects are observed.

M3 - Abstract

VL - 19

JO - Proceedings of the Physiological Society

JF - Proceedings of the Physiological Society

SN - 1749-6187

M1 - C120

ER -