Vesicle‐Released Glutamate Maintains Muscle Spindle Afferent Excitability in Adult Mice

Abstract

The molecular mechanisms by which Group Ia and II muscle spindle (MS) afferents transduce muscle movement into action potentials is not fully understood, although the rapidly adapting PIEZO2 mechanically sensitive cation channel is essential. Mechanosensation in many types of sensory neurons relies on PIEZO2, suggesting that unique response properties are mediated by the complement of molecular mediators found in the different sensory neurons. Synaptic-like vesicles containing glutamate are released from the MS afferent receptor ending in a stretch and calcium dependent manner, suggesting a way to couple depolarization and increased intracellular calcium via PIEZO2 with additional depolarizing current via glutamate. Here we test the hypothesis that vesicle-released glutamate is necessary for maintaining MS afferent excitability especially during the static phase of stretch. We isolated the extensor digitorum longus muscle and the sciatic nerve from adult mice (8-12 wks old) and compared identified MS afferent firing rates during ramp-and-hold stretch and vibration before and after the addition of glutamate and an inhibitor of glutamate packaging into vesicles (xanthurenic acid; XA). Glutamate addition led to a significant increase in MS afferent firing rate during the end of stretch (1mM; n=12 in control and glutamate groups), while blocking glutamate release with XA significantly decreased firing (3mM; n=17), with 5 afferents exhibiting a complete absence of firing. Firing rates during the dynamic phase of stretch and sinusoidal vibrations were not as affected by drug treatment. However, we noticed heterogeneity in MS afferent responses, with some afferents not responding to the drug treatments. To confirm our pharmacological findings, we used a transgenic mouse line with 1 or 2 copies of the vesicular glutamate 1 transporter gene (VGLUT1; B6.129X1-Sic17a7tm1Edw/MmcD; Fremeau, 2004). VGLUT1+/- afferents (n=14) had significantly lower firing rates during the end of stretch than afferents from wildtype littermates (WT; n=14), however firing rates during vibration were not significantly different. Importantly, 3 of 14 VGLUT1+/- afferents could not maintain firing throughout the entire 4 s stretch, something never seen in WT afferents. These results support our hypothesis that glutamate is critical to maintaining MS afferent firing during maintained stretch. Future studies will explore which glutamate receptor subtype and signaling pathway mediates this effect. A better understanding of MS afferent mechanotransduction could define targets for therapeutic intervention during diseases with abnormal afferent excitability.