Glutamatergic modulation of vesicle turnover in primary mechanosensory endings

Primary mechanosensory nerve terminals in vertebrates contain clear synaptic-like vesicles (SLVs). Previously autogenic modulation of mechanoreceptor excitability by glutamate release from SLVs has been demonstrated (Bewick et al, 2005) using Ia afferent primary endings of rat muscle spindle as a model. Our aim here was to test whether SLVs in other endings are functionally similar, using mechanosensitive lanceolate endings of mouse hair follicles. FM1-43 was used to characterise SLV recycling in lanceolate endings. C57/B16J mice (20 - 28 g, either sex) were killed by Schedule 1 methods, ASPA (1986). The inner and outer layers of ear skin were separated, cleaned and equilibrated in carboxygenated physiological solution (1 hr, 30°C). After labelling (30 min, 10 µM) in saline or test solution, FM1-43 was washed off (30 min) and then chelated (1 mM ADVASEP-7, 5 min). Fluorescent microscope images were saved on computer hard drive. Differences in net fluorescence intensity were compared (3-4 ears per group) for control (n = 33-40 terminals) and treated preparations by Student’s t-test, with a significance threshold of P < 0.05. Consistent with previous studies (Kain & Slater, 2003) 1 mM neomycin and 10 mM Ca2+ did not block FM1-43 uptake (n = 32; P = 0.117). Labelling was reversible, as terminals destained spontaneously. The dye release mechanism was tested with 3 nM α-latrotoxin, which elicits uncontrolled exocytosis. Latrotoxin markedly increased loss (52% at 15 min, n = 7, P < 0.0001; 70% at 30 min, n = 7, P < 0.0001) of label from terminals, indicating dye loss was predominantly through exocytosis. These data suggest dye fluxes were through SLV recycling, not mechanically sensitive channels. However, 3 mM Co2+ and 5 mM Mg2+ blocked FM1-43 labelling by 91.3% (n = 43; P < 0.001) and 71.5% (n = 39; P < 0.001), suggesting SLV recycling is Ca sensitive. Labelling was sensitive to glutamate since 1 mM doubled net terminal intensity (n = 34; P < 0.001). Conversely, 10 µM PCCG-13, a specific blocker of the non-canonical phospholipase D mGlu receptor, decreased dye uptake by 75% (n = 34; P < 0.001). 100 nM 5-fluoro-2-indolyldes-chlorohalopemide (FIPI) a new, highly specific PLD inhibitor (Monovich et al., 2007) also reduced net intensity by > 33% (n = 42; P < 0.01). These data suggest SLV recycling in lanceolate nerve endings and muscle spindle afferents share similar functional characteristics, and is sensitive to glutamate. This is consistent with a general role for SLVs in glutamatergic modulation of mechanosensation.