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. 2021 Jun;599(11):2953-2967.
doi: 10.1113/JP281182. Epub 2021 Apr 18.

Vesicle-released glutamate is necessary to maintain muscle spindle afferent excitability but not dynamic sensitivity in adult mice

Kimberly Than  1 , Enoch Kim  1 , Cebrina Navarro  1 , Sarah Chu  1 , Nikola Klier  1 , Alyssa Occiano  1 , Serena Ortiz  1 , Alexandra Salazar  1 , Steven R Valdespino  1 , Natanya K Villegas  1 , Katherine A Wilkinson  1
Affiliations

Vesicle-released glutamate is necessary to maintain muscle spindle afferent excitability but not dynamic sensitivity in adult mice

Kimberly Than et al. J Physiol. 2021 Jun.

Abstract

Key points: Muscle spindle afferents are slowly adapting low threshold mechanoreceptors that report muscle length and movement information critical for motor control and proprioception. 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. 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. 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.

Abstract: Muscle spindle afferents are slowly adapting low threshold mechanoreceptors that have both dynamic and static sensitivity to muscle stretch. The exact mechanism by which these neurons translate muscle movement into action potentials is not well understood, although the PIEZO2 mechanically sensitive cation channel is essential for stretch sensitivity. PIEZO2 is rapidly adapting, suggesting the requirement for additional molecular elements to maintain firing during stretch. Spindle afferent sensory endings contain glutamate-filled synaptic-like vesicles that are released in a stretch- and calcium-dependent manner. Previous work has shown that glutamate can increase and a phospholipase-D coupled metabotropic glutamate antagonist can abolish firing during static stretch. Here, we test the hypothesis that vesicle-released glutamate is necessary for maintaining muscle spindle afferent excitability during static but not dynamic stretch. To test this hypothesis, we used a mouse muscle-nerve ex vivo preparation to measure identified muscle spindle afferent responses to stretch and vibration. In C57BL/6 adult mice, bath applied glutamate significantly increased the firing rate during the plateau phase of stretch but not during the dynamic phase of stretch. Blocking the packaging of glutamate into vesicles by the sole vesicular glutamate transporter, VGLUT1, either with xanthurenic acid or by using a transgenic mouse with only one copy of the VGLUT1 gene (VGLUT1+/- ), decreased muscle spindle afferent firing during sustained stretch but not during vibration. Our results suggest a model of mechanotransduction where calcium entering the PIEZO2 channel can cause the release of glutamate from synaptic-like vesicles, which then helps to maintain afferent depolarization and firing.

Keywords: glutamate; mechanotransduction; muscle afferent; muscle spindle; sensory physiology.

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Conflict of interest statement

Competing Interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Glutamate is necessary for maintained firing during stretch.
A. Representative raw neural response before and after 1 mM glutamate addition during ramp-and-hold stretch. Instantaneous frequency of muscle spindle afferent firing before (black) and after glutamate (Glu; blue) shown overlaid below. Similar representative traces following 3 mM xanthurenic acid (XA; red) that caused a reduction in firing during stretch (B; unit XA1 in Fig. 2B as well) or a complete elimination by the end of the plateau phase of stretch (C; unit XA2 in Fig. 2C as well). Time course of the response at the beginning (Initial Static Time or IST; D) or end of stretch (Final Static Time or FST; G) and the maximum firing rate during ramp phase (Dynamic Peak or DP; J) shown for no drug controls (Ctrl; black solid line; n=12), 1 mM Glu (blue dashed line; n=12) or 3 mM XA (red dotted line; n=17). Time when drug was added shown in gray bar. All values expressed as the % of the average response during the 20 min baseline (%BL). Error bars denote ± standard deviation. Maximum increases in firing during the 40 min drug exposure shown as individual points for Ctrl, Glu and kainic acid (KA) during IST (E), FST (H), and DP (K). Individual points for maximum decreases in firing for Ctrl and XA during IST (F), FST (I), and DP (L). Gray lines denote the median and * denotes IST p=0.006, Independent Samples t-Test and FST p=0.005 Mann-Whitney U test.
Figure 2.
Figure 2.. Glutamate is not important for mediating dynamic sensitivity.
A. Representative raw neural response to 1 mM glutamate (Glu) in an afferent that could not entrain to 50 Hz vibration. Instantaneous frequency of muscle spindle afferent firing before (black) and after 1 mM glutamate (Glu; blue) shown overlaid below. All 4 afferents that could entrain to vibration before Glu continued to entrain post-Glu. Similar representative traces following 3 mM xanthurenic acid (XA; red) that caused a reduction in firing during stretch but no change in the ability to entrain to 100 Hz (B; same unit as Fig. 1B) and a unit that ceased firing during the static phase of stretch following XA but could still entrain to 50 Hz vibration (C; same unit as Fig. 1C). Example of 1 of 2 units following XA that eliminated firing during both ramp-and-hold stretch and vibration (D). Time course of the change in average firing rate during 50 Hz (E) or 100 Hz vibrations (H) shown for no drug controls (Ctrl; black solid line; n=11), 1 mM Glu (blue dashed line; n=11) and 3 mM XA (red dotted line; n=17). Time when drug was added shown in gray bar. All values expressed as the % of the average response during the 20 min baseline (%BL). Error bars denote ± standard deviation. Maximum increases in firing during the 40 min drug exposure shown as individual points for Ctrl and Glu during 50 Hz (F) and 100 Hz vibrations (I). Individual points for maximum decreases in firing for Ctrl and XA during 50 Hz (G) and 100 Hz vibrations (J). Gray lines denote the median. * denotes 50 Hz Average Firing Frequency p=0.023, Mann-Whitney U test and 100 Hz Average Firing Frequency p=0.013, Independent samples t-test.
Figure 3.
Figure 3.. Muscle spindle afferents of VGLUT1+/− mice have lower firing frequencies during static phase of stretch.
Characteristic responses of muscle spindle afferents isolated from WT mice to static stretch (A & B). Normal stretch response in firing pattern of VGLUT1+/− mice during the ramp-and-hold stretch seen in 11 of 14 afferents (C). Three out of 14 afferents of VGLUT1+/− mice failed to consistently fire throughout stretch (D; afferent VGLUT1+/−1 shown in red and VGLUT1+/−2 shown in black; E afferent VGLUT1+/−3; same afferents shown in Fig. 4B). Boxplots of firing rates of muscle spindle afferents during IST (F) and FST (G) at all 3 stretch lengths. Line represents group median and bars represent maximum and minimum group values. Differences in average firing rates between the WT (black; n = 14) and VGLUT1+/− afferents (red; n = 14) during IST and FST significantly different as determined by a two-way ANOVA (main effect of genotype, IST p = 0.011 and FST p = 0.10). (H) Difference in firing at FST at the 7.5% Lo stretch and that of the 2.5% Lo stretch were compared between genotypes. The black line represents the mean for a given group. No significant group differences observed (p = 0.194; Mann-Whitney UTest), but 4 VGLUT1+/− afferents had values of 0 or less, something never observed in WT afferents.
Figure 4.
Figure 4.. There is no difference in response to vibration in VGLUT1+/− afferents.
Most afferents of both genotypes had fairly normal responses to vibration (A). Top trace is raw neural firing of a typical WT (black) and bottom trace a VGLUT1+/− (red) muscle spindle afferent to a 50 μm/100 Hz vibration (length change on bottom). Firing patterns of the 3 muscle spindle afferents of VGLUT1+/− mice that failed to fire consistently throughout ramp-and-hold stretches (B; afferent labels the same as those used in raw traces Fig 3D-E) to a 25 μm/50 Hz vibration. 2 of 3 afferents responded at levels at or above the average change in firing rate for WT afferents. The third unit, VGLUT1+/−2, did not fire at all during vibration. Individual average firing rates in response to 25 μm (C) or 50 μm (D) vibrations of 4 amplitudes (x axis). WT afferents (n=14) are shown by filled black circles. VGLUT1+/− afferents shown in open red circles. Filled red circles denote the response of the 3 afferents that did not maintain firing during static stretch. Gray lines denote group median. Data from 5 μm and 100 μm vibrations not shown, but followed a similar pattern. There was no effect of genotype on average firing frequency during vibration (3 factor ANOVA, main effect of genotype p= 0.904).
Figure 5.
Figure 5.. Potential model for muscle spindle afferent mechanotransduction.
Schematic of the muscle spindle (top) and potential signaling pathway for synaptic-like vesicle released glutamate (bottom; modified from (Bewick et al., 2005)). Muscle stretch opens PIEZO2 which allows both Na+ and Ca++ entry into the cell and is likely one of the primary drivers of membrane depolarization during the dynamic phase of stretch. Ca++ can cause the fusion of glutamate containing synaptic-like vesicles and the released glutamate likely binds to a receptor on the afferent membrane. Potential candidates are a phospholipase-D coupled metabotropic glutamate receptor (PLD mGluR; (Bewick et al., 2005)) or metabotropic glutamate receptor 5 (mGluR5; (Lund et al., 2010)). Glutamate binding leads to additional depolarizing current potentially by increasing the activity of voltage-gated sodium channels, possibly NaV1.6, NaV1.7, and/or NaV1.1, which are all located in the afferent endings (Carrasco et al., 2017). Alternatively, glutamate increases depolarization via another unidentified mechanism. Figure created using BioRender.com.
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