Impact of Aging on Proprioceptive Sensory Neurons and Intrafusal Muscle Fibers in Mice

Abstract

The impact of aging on proprioceptive sensory neurons and intrafusal muscle fibers (IMFs) remains largely unexplored despite the central function these cells play in modulating voluntary movements. Here, we show that proprioceptive sensory neurons undergo deleterious morphological changes in middle age (11- to 13-month-old) and old (15- to 21-month-old) mice. In the extensor digitorum longus and soleus muscles of middle age and old mice, there is a significant increase in the number of Ia afferents with large swellings that fail to properly wrap around IMFs compared with young adult (2- to 4-month-old) mice. Fewer II afferents were also found in the same muscles of middle age and old mice. Although these age-related changes in peripheral nerve endings were accompanied by degeneration of proprioceptive sensory neuron cell bodies in dorsal root ganglia (DRG), the morphology and number of IMFs remained unchanged. Our analysis also revealed normal levels of neurotrophin 3 (NT3) but dysregulated expression of the tyrosine kinase receptor C (TrkC) in aged muscles and DRGs, respectively. These results show that proprioceptive sensory neurons degenerate prior to atrophy of IMFs during aging, and in the presence of the NT3/TrkC signaling axis.

Keywords: Degeneration; Dorsal root ganglia; Ia/II afferents; Motor function; Muscle spindle.

Figure 1.

Age-related changes in Ia afferents in the extensor digitorum longus (EDL) muscle. Ia afferents were examined in whole-mounted EDL muscles from 2-, 11-, and 15-month-old mice expressing YFP in all peripheral axons (A–C). Coil distance (CD in A; left arrow) and coil width (CW in A; right arrow) were measured in Ia afferents at muscle spindles. In 11- and 15-month-old mice, the coil distance and width were significantly increased compared with 2-month-old mice (D,E). Fewer spirals are found properly wrapping around the equatorial region of intrafusal muscle fibers, defined as unravelling (F), in older mice. Degeneration of the nerve endings was also associated with an increased incidence of axons with large blebs (G) in aged mice. Blebbing is indicated by the arrows in B and C. At least three animals per age group and at least five nerve endings per muscle were analyzed. Scale bar = 50 µm. Error bar = STE. *p < .05, **p < .01, ***p < .001.

Figure 2.

Age-related changes in Ia afferents in the soleus muscle. Ia afferents expressing YFP were examined in whole-mounted soleus muscles from the same animals used to analyze extensor digitorum longus muscles in Figure 1. The coil distance (A) and width (B) significantly increased in older soleus muscles. The incidence of Ia afferents unravelling from the equatorial region of intrafusal muscle fibers was also higher in older mice (C). Sensory axons with large blebs were more numerous in old mice (D). At least three animals per age group and at least five nerve endings per muscle were analyzed. Error bar = STE. *p < .05, **p < .01, ***p < .001.

Figure 3.

Loss of II afferents in the extensor digitorum longus (EDL) and soleus muscles of middle age and old mice. Proprioceptive sensory nerve endings were visualized using transgenic mice expressing YFP in neurons. Type Ia afferents (A; right arrow) form primary muscle spindles whereas type II afferents (A; left arrow) form secondary muscle spindles. In older mice, there were fewer II afferents innervating muscle spindles (MS) in the EDL (C) and soleus (D) muscles. However, the number of type Ia afferents was unchanged in both muscles in aged mice (C,D). At least three animals were analyzed per age group and at least five nerve endings per muscle. PSA = proprioceptive sensory afferent. Scale bar = 50 µm. Error bar = STE. *p < .05, **p < .01, ***p < .001.

Figure 4.

Age-related changes in α-motor axons parallel those found in sensory afferents. Analysis of α-motor axons (YFP) innervating the postsynaptic site on extrafusal muscle fibers, visualized by using fluorescently tagged α-bungarotoxin (BTX). In the same muscles used to examine sensory afferents, fewer α-motor axons were found fully innervating postsynaptic sites in older mice (A–D). At least three animals per age group and at least 50 NMJs per muscle were analyzed. Scale bar = 20 µm. Error bar = STE. *p < .05, **p < .01, ***p < .001.

Figure 5.

The number and size of intrafusal muscle fibers (IMFs) are unchanged in older mice. Nuclear bag fibers were stained using an antibody against S46, which binds to slow-tonic myosin heavy chain isoform (A–C). The perimeter of all muscle fibers was labeled with an antibody against laminin (A–C). We examined the tibialis anterior muscle of 2-month (A), 11-month (B), and 17-month-old (C) animals. The number of IMFs per spindle is similar between young and older mice (D). Aging did not affect the size of S46-positive and S46-negative IMFs (E). S46-positive IMFs do not differ between mice of different age groups (F). At least three animals per age group and at least 30 muscle fibers per muscle were analyzed. Scale bar = 50 µm. Error bar = STE. *p < .05, **p < .01, ***p < .001.

Figure 6.

The size of extrafusal muscle fibers decreases with aging. The perimeter of extrafusal muscle fibers in the tibialis anterior muscle was revealed by immunostaining for laminin in young and old mice (A,B). In 17-month-old mice, there is a significant reduction in the size of extrafusal muscle fibers compared with those in 2-month-old mice (C). At least three animals per age group and 30 muscle fibers per animal were analyzed. Scale bar = 50 µm. Error bar = STE. **p < .01.

Figure 7.

Aging causes complete degeneration of sensory axons innervating the extensor digitorum longus (EDL) muscle in middle age and old mice. Sensory axons innervating the portion of the EDL muscle that controls the 2nd digit were analyzed using transgenic mice expressing YFP in neurons (A–C). Axon number was determined by digitally slicing through the 3D image plane (A′–C′). Fewer sensory axons innervating the EDL muscle were found in 11- and 15-month-old mice (D). Aged sensory axons also often exhibited deleterious structural changes that include thinning and large swellings. At least three animals per age group were analyzed. Scale bar = 10 µm. Error bar = STE. ***p < .001.

Figure 8.

Fewer proprioceptive sensory neurons are present in middle age and old DRGs. Proprioceptive sensory neurons (PSNs) selectively expressing YFP within DRGs were whole mounted and visualized using confocal microscopy (A–C). Analysis of L3 DRGs revealed that PSNs are significantly reduced in 11- and 17-month-old mice compared with 2-month-old mice (D). Tyrosine kinase receptor C (TrkC) transcripts are reduced in 13-month-old but then significantly increase in 21-month-old DRGs (E) whereas neurotrophin 3 (NT3) transcripts remain unchanged in the tibialis anterior muscle (F) compared with 4-month-old mice. At least three animals per age group were analyzed. Scale bar = 150 µm. Error bar = STE. *p < .05, **p < .01.