Differential encoding of mammalian proprioception by voltage-gated sodium channels

Abstract

Animals that require purposeful movement for survival are endowed with mechanosensory neurons called proprioceptors that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we have identified distinct and nonredundant roles for two voltage-gated sodium channels (Na_Vs), Na_V1.1 and Na_V1.6, in mammalian proprioception. Deletion of Na_V1.6 in somatosensory neurons (Na_V1.6^cKO mice) causes severe motor deficits accompanied by complete loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target Na_V1.1 (Na_V1.1^cKO). In Na_V1.6^cKO animals, loss of proprioceptive feedback caused non-cell-autonomous impairments in proprioceptor end-organs and skeletal muscle that were absent in Na_V1.1^cKO mice. We attribute the differential contribution of Na_V1.1 and Na_V1.6 in proprioceptor function to distinct cellular localization patterns. Collectively, these data provide the first evidence that Na_V subtypes uniquely shape neurotransmission within a somatosensory modality.

Fig. 1.. Na_V1.6 is required for somatosensory neuron-driven motor behaviors and function.

Representative images showing limb position of adult Na_V1.6^fl/fl (A), Na_V1.6^het (B), and Na_V1.6^cKO (C) mice suspended from the tail (above) and on flat surface (below). White arrows indicate the direction of hind limbs. Quantification of distance traveled (D, Na_V1.6^het p > 0.999 , Na_V1.6^cKO p = 0.0003, compared to Na_V1.6^fl/fl), average speed (E, Na_V1.6^het p > 0.999 , Na_V1.6^cKO p = 0.0003), and the percent of time spent moving (F, Na_V1.6^het p > 0.999, Na_V1.6^cKO p = 0.05, compared to Na_V1.6^fl/fl) for Na_V1.6^fl/fl (cyan), Na_V1.6^het (grey), and Na_V1.6^cKO (magenta) mice as measured in the open field assay for a 10-minute testing period. (G) Average grip force in grams measured across 6 consecutive trials; Na_V1.6^het p = 0.4947, Na_V1.6^cKO p < 0.0001, compared to Na_V1.6^fl/fl. (H) Average latency to fall from the rotarod across three consecutive training days. No statistically significant genotype-dependent difference was observed (p = 0.1342). (I) Average latency to fall on third day of testing (Na_V1.6^het p = 0.3943, compared to Na_V1.6^fl/fl). Each dot represents one animal, except in (H) were each dot represents the mean across animals. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. A Kruskal-Wallis test with Dunn’s multiple comparisons (D to G), a Two-way ANOVA with Sidak’s multiple comparisons (H), and a Welch’s T-test (I) were used to determine statistical significance. Na_V1.6^fl/fl N = 8, Na_V1.6^het N=20, Na_V1.6^cKO N=20.

Fig. 2.. Loss of Na_V1.6 abolishes muscle proprioceptor static stretch sensitivity.

Representative responses to ramp-and-hold muscle stretch at 7.5% of optimal length (Lo) in Na_V1.6^fl/fl (A), Na_V1.6^het (B), and Na_V1.6^cKO (C) muscle proprioceptors. The percentage of afferents that displayed resting discharge at Lo are represented by the pie charts to the right (black indicates absence of resting discharge). (D) Quantification of afferent firing frequency 3.25 to 3.75 seconds into stretch protocol. Na_V1.6^fl/fl (cyan), Na_V1.6^het (grey), and Na_V1.6^cKO (magenta). Na_V1.6^het p = 0.178, Na_V1.6^cKO p = 0.001, compared to Na_V1.6^fl/fl. (E) Firing regularity was quantified as the coefficient of variation of the interspike interval (ISI CV) 1.5 to 3.5 seconds into the stretch protocol. Na_V1.6^het p = 0.669, Na_V1.6^cKO p = 0.000, compared to Na_V1.6^fl/fl. In 6 out of 10 animals we observed no response to stretch and therefore could only include the quantifiable responses from 4 afferents from Na_V1.6^cKO mice. Only quantifiable responses were included in statistical analyses in D and E. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. Each dot represents a single afferent. A two-way mixed-design ANOVA (Dunnett’s post-hoc comparison) was used to determine statistical significance in D and E. Na_V1.6^fl/fl n = 8, N=7; Na_V1.6^het n=8, N=8; Na_V1.6^cKO n=4, N=10. n = afferents, N = mice.

Fig 3.. Na_V1.6 is required for proprioceptor responses to vibration.

Representative traces from Na_V1.6^fl/fl (A), Na_V1.6^het (B), and Na_V1.6^cKO (C) afferents that were able to entrain to a 25 Hz, 100 μm amplitude vibration stimulus. Tables to the right indicate the percentage of afferents that were able to entrain across stimulus frequencies and amplitudes (Na_V1.6^fl/fl, top; Na_V1.6^het, middle; Na_V1.6^cKO, bottom; D to E) Quantification of firing frequency across vibration amplitudes. At 25μm (D) Na_V1.6^het p = 0.005 (# denotes significance in Na_V1.6^het), Na_V1.6^cKO p = 0.001, compared to Na_V1.6^fl/fl (* denotes significance in Na_V1.6^cKO). At 50μm (E) Na_V1.6^het p = 0.053, Na_V1.6^cKO p = 0.002, compared to Na_V1.6^fl/fl. At 100μm (F) Na_V1.6^het p = 0.414, Na_V1.6^cKO p = 0.018 compared to Na_V1.6^fl/fl. Na_V1.6^fl/fl (cyan), Na_V1.6^het (grey), and Na_V1.6^cKO (magenta). A two-way mixed-design ANOVA (Dunnett’s post-hoc comparison) was used to determine statistical in D to F. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. Each dot represents the average afferent response per genotype. Na_V1.6^fl/fl n = 8, N=7; Na_V1.6^het n=8, N=8; and Na_V1.6^cKO n= 4, N=10. n=afferents, N=mice.

Fig. 4.. Na_V1.6 plays a developmentally dependent role in proprioceptor synaptic transmission in the spinal cord.

(A) Representative monosynaptic reflex responses from Na_V1.6^fl/fl (cyan), Na_V1.6^het (grey), and Na_V1.6^cKO (magenta) hemicords during postnatal days 6 to 11. Quantification of response properties. (B) Response latency, Na_V1.6^het p = 0.760, Na_V1.6^cKO p = 0.019, compared to Na_V1.6^fl/fl. (C) Monosynaptic response amplitude, Na_V1.6^het p = 0.238 , Na_V1.6^cKO p = 0.640, compared to Na_V1.6^fl/fl. (D) Stimulus threshold, Na_V1.6^het p = 0.910, Na_V1.6^cKO p = 0.271, compared to Na_V1.6^fl/fl. (E) Full width half max, Na_V1.6^het p = 0.999, Na_V1.6^cKO p = 0.929, compared to Na_V1.6^fl/fl. (F) Polysynaptic response amplitude, Na_V1.6^het p = 0.514, Na_V1.6^cKO p = 0.704, compared to Na_V1.6^fl/fl. (G) Representative monosynaptic reflex responses in Na_V1.6^fl/fl (cyan), Na_V1.6^het (grey), and Na_V1.6^cKO (magenta) hemicords during postnatal days 14 to 18. (H) Response latency, Na_V1.6^het p = 0.023, Na_V1.6^cKO p < 0.0001, compared to Na_V1.6^fl/fl. (I) Monosynaptic response amplitude, Na_V1.6^het p = 0.037, Na_V1.6^cKO p = 0.018, compared to Na_V1.6^fl/fl. (J) Stimulus threshold, Na_V1.6^het p = 0.164, Na_V1.6^cKO p <0.0001 , compared to Na_V1.6^fl/fl. (K) Full width half max, Na_V1.6^het p = 0.784, Na_V1.6^cKO p <0.0001 , compared to Na_V1.6^fl/fl. (L) Polysynaptic response amplitude, Na_V1.6^het p = 0.143, Na_V1.6^cKO p = 0.092, compared to Na_V1.6^fl/fl. Each dot represents a single hemicord. (A to F) Na_V1.6^fl/fl n=9, Na_V1.6^het n=13, and Na_V1.6^cKO n=14. (G to L) Na_V1.6^fl/fl n=8, Na_V1.6^het n=12, and Na_V1.6^cKO n=15. N=8–15. n=hemicords, N=mice. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. A two-way mixed-design ANOVA (Tukey’s post-hoc comparison) was used to determine statistical significance.

Fig. 5.. Na_V1.1 does not contribute to proprioceptor synaptic transmission prior to the onset of walking behaviors.

(A) Representative monosynaptic reflex responses from Na_V1.1^fl/fl (cyan), Na_V1.1^het (grey), and Na_V1.1^cKO (magenta) hemicords recorded during postnatal days 6 to 11. (B to D) Quantification of monosynaptic response properties. (B) Response latency, Na_V1.1^het p = 0.723, Na_V1.1^cKO p = 0.238, compared to Na_V1.1^fl/fl. (C) Monosynaptic response amplitude, Na_V1.1^het p = 0.378, Na_V1.1^cKO p > 0.999, compared to Na_V1.1^fl/fl. (D) Stimulus threshold, Na_V1.1^het p>0.999 , Na_V1.1^cKO p>0.999 , compared to Na_V1.1^fl/fl. (E) Full width half max, Na_V1.1^het p>0.999 , Na_V1.1^cKO p = 0.255, compared to Na_V1.1^fl/fl. (F) Polysynaptic response amplitude, Na_V1.1^het p = 0.574, Na_V1.1^cKO p = 0.473, compared to Na_V1.1^fl/fl. Each dot represents a single hemicord. Na_V1.1^fl/fl n=12, Na_V1.1^het n=4, and Na_V1.1^cKO n=9. N=4–10 mice. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. A Kruskal-Wallis test with Dunn’s multiple comparisons was used to determine statistical significance.

Fig. 6.. Electrical signaling deficits associated with Na_V1.6- but not Na_V1.1-deletion impairs muscle spindle development.

Representative confocal images of muscle spindles from (A) Na_V1.6^fl/fl, (B) Na_V1.6^het, and (C) Na_V1.6^cKO extensor digitorum longus (EDL) muscle sections (30 μm). Images were acquired with a 60x oil 1.4 NA lens. VGLUT1 (grey scale) labels proprioceptor sensory terminals and DAPI (cyan) labels nuclei. Insets below images show the colocalization of sensory terminals with DAPI. (D) Quantification of wrapping efficiency index based on colocalization of DAPI with VGLUT1. Na_V1.6^het p = 0.0658, and Na_V1.6^cKO, p < 0.0001, compared to Na_V1.6^fl/fl. Representative images of muscle spindles from (E) Na_V1.1^fl/fl, (F) Na_V1.1^het, (G) Na_V1.1^cKO. (H) Quantification of wrapping efficiency index based on colocalization of DAPI with VGLUT1. Na_V1.1^het, p = 0.762, and Na_V1.1^cKO, p = 0.282, compared to Na_V1.1^fl/fl. Each dot represents a single muscle spindle section. (D) Na_V1.6^fl/fl n=8, Na_V1.6^het n=12, and Na_V1.6^cKO n=10. (H) Na_V1.1^fl/fl n=10, Na_V1.1^het n=8, and Na_V1.1^cKO n=14. N=3 mice per genotype. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. A one-way ANOVA (Dunnett’s post-hoc comparison) was used to determine statistical significance. Scale bar = 20 μm. Inset scale bar = 10 μm.

Fig 7.. Loss of proprioceptive feedback alters skeletal muscle development in Na_V1.6^cKO mice.

Representative images of muscle fibers from the soleus of (A) Na_V1.6^fl/fl, (B) Na_V1.6^het, and (C) Na_V1.6^cKO mice. Images were acquired with a 20× 0.75 NA air lens. Myosin heavy chain (MHC), labels slow twitch muscle fibers (cyan), MHC type IIa labels fast twitch muscle fibers (magenta), and wheat germ agglutinin (WGA, yellow) labels the cell membrane of muscle fibers. (D and E) Quantification of muscle fiber anatomy. (D) Fiber area, Na_V1.6^het p = 0.337, Na_V1.6^cKO p = 0.006, compared to Na_V1.6^fl/fl. (E) cumulative distribution plots showing the muscle fiber area in the soleus between Na_V1.6^fl/fl (cyan), Na_V1.6^het (grey), and Na_V1.6^cKO (magenta) mice. (F to I) Quantification of intrinsic properties of soleus muscle. (F) Tetanus stress, Na_V1.6^het p = 0.926, Na_V1.6^cKO p = 0.990, compared to Na_V1.6^fl/fl. (G) Tetanus force, Na_V1.6^het p = 0.925, Na_V1.6^cKO p = 0.690, compared to Na_V1.6^fl/fl. (H) Percentage of force post-fatigue, Na_V1.6^het p = 0.189, Na_V1.6^cKO p = 0.150, compared to Na_V1.6^fl/fl. (I) Percentage of force post-recovery, Na_V1.6^het p = 0.851, Na_V1.6^cKO p = 0.293, compared to Na_V1.6^fl/fl. Each dot represents a single animal. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. A one-way ANOVA (Dunnett’s post-hoc comparison) was used to determine statistical significance. Scale bar=50 μm.

Fig. 8.. Mildly impaired proprioceptive feedback does not impair skeletal muscle development.

Images of muscle fibers from (A) Na_V1.1^fl/fl, (B) Na_V1.1^het, and (C) Na_V1.6^cKO soleus muscle. Images were acquired with a 20× 0.75 NA air lens. Myosin heavy chain (MHC) labels slow twitch muscle fibers (cyan), MHC type IIa labels fast twitch muscle fibers (magenta), and wheat germ agglutinin (WGA, yellow) labels the cell membrane of muscle fibers. (D and E) Quantification of muscle fiber anatomy. (D) Fiber area, Na_V1.1^het p = 0.9827, Na_V1.1^cKO p = 0.880, compared to Na_V1.1^fl/fl. (E) cumulative distribution plots showing the muscle fiber area between Na_V1.1^fl/fl (cyan), Na_V1.1^het (grey), and Na_V1.1^cKO (magenta). (F to I) Quantification of intrinsic properties of soleus muscle. (F) Tetanus stress, Na_V1.1^het p = 0.841, Na_V1.1^cKO p = 0.596, compared to Na_V1.1^fl/fl. (G) Tetanus force, Na_V1.1^het p = 0.624, Na_V1.1^cKO p = 0.978, compared to Na_V1.1^fl/fl. (H) Percentage of force post-fatigue, Na_V1.1^het p = 0.999, Na_V1.1^cKO p = 0.934, compared to Na_V1.1^fl/fl. (I) Percentage of force post-recovery, Na_V1.1^het p = 0.724, Na_V1.1^cKO p = 0.993, compared to Na_V1.1^fl/fl. Each dot represents a single animal. Box and whisker plots represent maximum, minimum, median, upper and lower quartiles of data sets. A one-way ANOVA (Dunnett’s post-hoc comparison) was used to determine statistical significance. Scale bar=50 μm.

Fig 9.. Na_V1.1 and Na_V1.6 localize to discrete cellular regions in muscle spindles.

VGLUT1 (magenta, A) labeled muscle spindles express clusters of Na_V1.6 (cyan, B and C). Arrows denote clusters of Na_V1.6. (D) Quantification of the number of Na_V1.6 clusters per muscle spindle (n=15 spindles). (E to H) Na_V1.6 clusters (G) colocalize with Ankyrin-G (yellow, E and F are insets from G). (H) Quantification of the percentage of Ankyrin G clusters that colocalize with Na_V1.6 clusters (n=9 spindles). (I to M) Co-labeling of Na_V1.6 (J) with juxtaparanode maker CASPR (K, yellow) reveal proprioceptor nodes of Ranvier (L) and heminodes (M) (n=10 spindles). Nodes of Ranvier were identified by two CASPR+ signals (arrows) flanking Na_V1.6 clusters (arrowhead). Heminodes were identified by a single CASPR+ signals juxtaposed to Na_V1.6 cluster. (N to S) Co-labeling of Na_V1.6 (O) with Na_V1.1 (P, yellow) show discrete cellular expression patterns. (Q to S) Arrowhead denotes Na_V1.6 channels and arrows denote Na_V1.1 channels. N=3–5 mice. Inset scale bars=10 μm. Scale bars=20 μm

Fig. 10.. Model of proprioceptive transmission by Na_V1.1 and Na_V1.6.

Upon muscle stretch, Piezo2 (red) transduces mechanical stimuli into electrical potentials. Following Piezo2 activation, Na_V1.1 (blue) expressed in muscle spindle sensory terminals drives consistent proprioceptor firing during static muscle stretch. Na_V1.6 (yellow) localized to heminodes and nodes of Ranvier initiate and propagate all proprioceptive signals from muscle spindles to spinal cord. It is likely prior to walking, there is functional redundancy of Na_Vs in proprioceptive axons. After walking behaviors emerge however, proprioceptive synaptic transmission is dependent on Na_V1.6. Deletion of Na_V1.6 in all sensory neurons led to a significant decrease in skeletal muscle fiber size that was not present in Na_V1.1^cKO muscle, suggesting that complete loss of proprioceptive feedback non-cell autonomously regulates skeletal muscle development.