Immunogold labelling for glutamate in lanceolate endings of rat hairs

50 nm diameter, clear synaptic-like vesicles (SLVs) are found in primary mechanosensory nerve terminals. Our studies of sensory endings of rat Ia afferents suggest SLVs are part of a glutamatergic system that modulates mechanoreceptor excitability; in particular, glutamate-like immunoreactivity indicated a high glutamate content in these endings (Bewick et al, 2005). We now show that lanceolate endings of rat hair follicles also have high levels of glutamate. Adult rats (3) were deeply anaesthetized with sodium pentobarbitone (45 mg kg−1, I.P.) and fixed by transcardial perfusion (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.4). Samples of pinna skin and cerebellum (positive control) were embedded in Araldite for post-embedding immunocytochemistry using rabbit polyclonal antibodies (Sigma) against glutamate or GABA. 10nm gold-labelled antibody (goat anti-rabbit IgG, BBInternational) was used for secondary incubation. EMgraphs (magnification 40000 to 60000x) were analysed with ImageJ (NIH, USA). Profiles of lanceolate endings and their accessory cells of skin; and mossy fibres, granule cells and Golgi cells of cerebellum were sampled for quantification. Glutamate-specific labelling was greater in lanceolate endings (mean 28.2 ± SE 1.73 gold particles/µm2, n = 84) than in the surrounding accessory cells (mean 9.3 ± SE 0.80 particles/µm2, n = 84; P ≤ 0.002, ANOVA and post hoc Tukey test), and was similar to that in mossy fibres (mean 28.2 ± SE 2.39 particles/µm2, n = 28) and granule cells (mean 22.6 ± SE 1.13 gold particles/µm2, n = 45). GABA-specific immunoreactivity was not found in either the lanceolate endings or accessory cells of the skin. However anti-GABA immunoreactivity was evident in the cerebellum, particularly in Golgi-cell axons. In conclusion these data support our hypothesis that regulation of mechanoreceptor function by glutamate release from SLVs may be a general modulatory mechanism, common to other mechanosensitive structures in addition to muscle spindles.