Glutamatergic modulation of synaptic‐like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles

Abstract

Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate ending would provide support for our hypothesis that it is a general property of fundamental importance to the regulation of the responsiveness of the broad class of primary mechanosensory endings. We show not only that an SLV-based system is present in lanceolate endings, but also that there are clear parallels between its operation in the two types of mechanosensory endings. In particular, we demonstrate that, as in the muscle spindle: (i) FM1-43 labels the sensory terminals of the lanceolate ending, rather than the closely associated accessory (glial) cells; (ii) the dye enters and leaves the terminals primarily by SLV recycling; (iii) the dye does not block the electrical response to mechanical stimulation, in contrast to its effect on the hair cell and dorsal root ganglion cells in culture; (iv) SLV recycling is Ca2+ sensitive; and (v) the sensory terminals are enriched in glutamate. Thus, in the lanceolate sensory ending SLV recycling is itself regulated, at least in part, by glutamate acting through a phospholipase D-coupled metabotropic glutamate receptor.

Morphology of P2X3‐immunoreactive nerve endings in the rat tracheal mucosa

“…Functionally, glutamate released from nerve endings in muscle spindles have been shown to regulate nerve endings themselves in order to maintain their excitability (Bewick, Reid, Richardson, & Banks, 2005). A similar glutamate-mediated autoregulation system was also suggested in lanceolate endings in sinus hairs (Banks et al, 2013) and Pacinian corpuscles (Pawson et al, 2009).…”

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

“…The dyes then label intracellular membranes, and this labeling is irreversible. However, in hair cells that are not mechanically stimulated 13,14 and in fully differentiated primary sensory nerve terminals in situ, such as Ia endings in muscle spindles 15 , and in the lanceolate endings here 7 , styryl dye labeling seems to reflect membrane endocytosis, since labeling is reversible and does not block the mechanosensory responses 7,15,16 . While some dye internalization by channel permeation in these endings cannot be ruled out completely, it is clear from the continued firing during dye incubation and the reversibility of the labeling that the great majority of the labeling in differentiated terminals in situ is by internalization with recycling vesicle membrane.…”

“…with Npy2r-linked GFP expression 17 . A final example may be investigating the role of synaptic-like vesicles (SLVs) 7 in these lanceolate terminals by examining the effect of modulators of SLV turnover (Ca, Mg, latrotoxin, glutamate receptor ligands) on stretch-evoked responses and styryl dye uptake/release. Thus, this new technique opens up a range of potentially interesting avenues of research in mechansensory neuroscience.…”

“…We have used the ear skin preparation to investigate the characteristics of the internalization and release of the fluorescent membrane styryl pridinium dyes 7 , a technique originally developed to study localized vesicle membrane recycling in synaptic terminals 8 . In synapses, imaging is also readily combined with simultaneous electrophysiological recording of responses in identified terminals 8,9 .…”

“…Pin the pinna skin to the PDMS-lined base of a recording chamber with fine insect pins around the edges (~6-8 per ear), placing the cleaned nerves at the base of the ear near two suction electrodes -one for recording (to take the nerve) and the other an indifferent electrode (to provide the neutral signal to a differential amplifier, see 7 ). 2.…”