Muscle spindle afferents are slowly adapting low threshold mechanoreceptors that report muscle length and movement information critical for motor control and proprioception.r The rapidly adapting cation channel PIEZO2 has been identified as necessary for muscle spindle afferent stretch sensitivity, although the properties of this channel suggest that additional molecular elements are necessary for mediating the complex slowly adapting response of muscle spindle afferents.r We report that glutamate increases muscle spindle afferent static sensitivity in an ex vivo mouse muscle nerve preparation, although blocking glutamate packaging into vesicles by the sole vesicular glutamate transporter, VGLUT1, either pharmacologically or by transgenic knockout of one allele of VGLUT1 decreases muscle spindle afferent static but not dynamic sensitivity.r Our results confirm that vesicle-released glutamate is an important contributor to maintained muscle spindle afferent excitability and may suggest a therapeutic target for normalizing muscle spindle afferent function.

The voltage-gated sodium channel (NaV), NaV1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for NaV1.1 in mammalian proprioception. RNAscope analysis and in vitro patch clamp recordings in genetically identified mouse proprioceptors show ubiquitous channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of NaV1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings from conditional knockout mice found that loss of NaV1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other NaV isoforms are sufficient to elicit activity in response to transient stimuli, NaV1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, NaV1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which NaV1.1 function is compromised. Collectively, we present the first evidence that NaV1.1 is essential for mammalian proprioceptive signaling and behaviors.

The mammalian voltage-gated sodium channel (NaV), NaV1.1, has been well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we report a new role for peripherally expressed NaV1.1 in murine motor behaviors. RNAscope analysis found 100% of proprioceptors express NaV1.1 transcript, consistent with in vitro patch clamp recordings showing this channel is required for repetitive firing in proprioceptors. Notably, genetic deletion of NaV1.1 in all sensory neurons caused profound motor coordination deficits in homozygous conditional knockout animals of both sexes, a phenotype similar to conditional Piezo2-knockout animals. Movement deficits were also observed in heterozygotes, demonstrating that NaV1.1 haploinsufficiency in sensory neurons leads to motor deficiencies. This behavioral phenotype was not due to reduced proprioceptor numbers or abnormal muscle spindle formation; however, we observed decreased proprioceptor innervation of motor neurons in the spinal cord in conditional knockouts, indicating loss of NaV1.1 in sensory neurons alters spinal cord circuitry. Ex vivo muscle afferent recordings also support the notion that loss of NaV1.1 leads to aberrant proprioceptor function. Collectively, these data provide the first evidence that NaV1.1 in mammalian sensory neurons is essential for motor coordination. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia. Thus, our data suggest sensory neuron dysfunction may contribute to the clinical manifestations and co-morbidities of neurological disorders in which NaV1.1 function is compromised.

The primary sensory input for proprioception, or the sense of body position in space, comes from muscle spindle afferents, which are mechanoreceptors that relay information about changes in muscle length. The muscle spindle is also innervated by sympathetic neurons, but the role of this sympathetic innervation on muscle spindle function is not well understood. Here we test the hypothesis that muscle spindle afferents will exhibit decreased firing in response to muscle stretch following exposure to the sympathetic neurotransmitters norepinephrine and epinephrine. To test this hypothesis we used an ex vivo mouse muscle‐nerve preparation. We recorded muscle spindle afferent firing activity during ramp‐and‐hold stretch and sinusoidal vibration before and after the addition of norepinephrine, epinephrine, or adrenergic receptor agonists. We observed significantly decreased firing during the end of stretch after both norepinephrine (100 µm, n=6) and epinephrine (30 µm, n=6). To identify the adrenergic receptor(s) involved, we also tested two ɑ2 adrenergic receptor agonists, Clonidine and Dexmedetomidine, and the ɑ1 receptor agonist phenylephrine. Both ɑ2 receptor agonists caused a decrease in muscle spindle afferent firing (1 mM clonidine, n=8 decreased firing; 100 µm dexmedetomidine, n=3). Phenylephrine had no significant effect on muscle spindle afferent firing (100 µm n=2; 30 µm n=6). These results show direct effects of sympathetic neurotransmitters on muscle spindle afferent stretch sensitivity and further support the hypothesis that sympathetic innervation of the muscle spindle plays an important role in modulating muscle spindle afferent activity and therefore motor control. Future studies will investigate the location of the adrenergic receptor(s) mediating this effect as well as the mechanism of action.

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.