Glutamate-enhanced muscle spindle excitability - inhibition by PLD-coupled metabotropic glutamate receptor antagonists

Guy Smith Bewick, R Duncan, W Eddie, C Maguire, Robert W Banks

Research output: Contribution to journalAbstract

Abstract

50nm, clear synaptic-like vesicles (SLVs) are abundant in primary afferent mechanosensory endings. In muscle spindle primary endings, SLVs appear glutamatergic, and undergo exo- and endocytosis at a rate modulated by mechanical activity (Banks et al. 2000; Bewick et al. 2000). Exogenous glutamate increases stretch-induced spindle discharge but antagonists of ionotropic (kynurenate) and most metabotropic (MCPG, group I&II; CPPG & MAP4, group III) glutamate receptors do not abolish this effect, even applied together. We have now used antagonists of phospholipase D-coupled mGlu (PLD-mGlu) receptors (Albani-Torregrossa et al. 1999) to try to block exogenous glutamate effects and probe for endogenous glutamate release. Adult rats (>300gm) were killed by Schedule 1 methods (Animals (Scientific Procedures) Act, 1986) and 4th lumbrical nerve-muscle preparations excised. Stretch-induced spindle discharge recordings from the muscle nerve with Ag wire electrodes were used to count spikes in the first 1.5s of the ‘hold’ phase of 1mm stretch-and-hold cycles (~10% increase in muscle length), avoiding the initial ‘dynamic’ response. Data are expressed as percentages (mean ± SE, of n preparations) of the pre-drug count and the significance of differences determined by paired t-test. 100µM glutamate increased afferent discharge (137.4 ± 14.4% vs pre-drug spike count, 4, P<0.05). As previously, MCPG (1mM) and CPPG (100nM) applied together were ineffective (156.8 ± 14.9%, 4, P<0.03), excluding a Group IIII mGlu receptor-mediated effect. We next tested for the involvement of PLD-mGlu receptors. This receptor is designated outside the standard Group I-III classification, and is inhibited by the Group I agonist DHPG. DHPG (200µM) application reduced the glutamate-mediated enhancement (111.3 ± 7.6%, 4, P>0.2). The effectiveness of the selective antagonist PCCG-13 (1µM) confirmed the involvement of PLD-coupled receptors (71.8 ± 8.6%, 6, P<0.05). To test for endogenous glutamate release (e.g. by exocytosis from glutamatergic SLVs) activating the same pathway, PCCG-13 was then applied without exogenous glutamate. 1µM PCCG-13 alone reduced mean excitability (81.2 ± 13.1%, 6), but this was not significant (P<0.1). However, 10µM PCCG-13 inhibited profoundly (24.7 ± 7.9%, 6, P<0.01). These data support the hypothesis that muscle spindle SLVs release glutamate, activating PLD-mGlu receptors and enhancing afferent discharge. The functional significance of this mechanism is unclear, but it appears crucial for regulating mechanosensory transduction by stretch-sensitive channels.
Original languageEnglish
Article numberC91
Number of pages1
JournalThe Journal of Physiology
Volume557P
Publication statusPublished - 2004

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