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.
|Journal||Proceedings of the Physiological Society|
|Publication status||Published - 2010|