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. 2018 Feb 7;119(5):1852–1862. doi: 10.1152/jn.00868.2017

Phrenic motor neuron loss in aged rats

Matthew J Fogarty 1,3, Tanya S Omar 1, Wen-Zhi Zhan 1, Carlos B Mantilla 1,2, Gary C Sieck 1,2,✉
PMCID: PMC6008083  PMID: 29412773

Abstract

Sarcopenia is the age-related reduction of muscle mass and specific force. In previous studies, we found that sarcopenia of the diaphragm muscle (DIAm) is evident by 24 mo of age in both rats and mice and is associated with selective atrophy of type IIx and IIb muscle fibers and a decrease in maximum specific force. These fiber type-specific effects of sarcopenia resemble those induced by DIAm denervation, leading us to hypothesize that sarcopenia is due to an age-related loss of phrenic motor neurons (PhMNs). To address this hypothesis, we determined the number of PhMNs in young (6 mo old) and old (24 mo old) Fischer 344 rats. Moreover, we determined age-related changes in the size of PhMNs, since larger PhMNs innervate type IIx and IIb DIAm fibers. The PhMN pool was retrogradely labeled and imaged with confocal microscopy to assess the number of PhMNs and the morphometry of PhMN soma and proximal dendrites. In older animals, there were 22% fewer PhMNs, a 19% decrease in somal surface area, and a 21% decrease in dendritic surface area compared with young Fischer 344 rats. The age-associated loss of PhMNs involved predominantly larger PhMNs. These results are consistent with an age-related denervation of larger, more fatigable DIAm motor units, which are required primarily for high-force airway clearance behaviors.

NEW & NOTEWORTHY Diaphragm muscle sarcopenia in rodent models is well described in the literature; however, the relationship between sarcopenia and frank phrenic motor neuron (MN) loss is unexplored in these models. We quantify a 22% loss of phrenic MNs in old (24 mo) compared with young (6 mo) Fischer 344 rats. We also report reductions in phrenic MN somal and proximal dendritic morphology that relate to decreased MN heterogeneity in old compared with young Fischer 344 rats.

Keywords: neuro-motor control, phrenic motor neuron, sarcopenia

INTRODUCTION

Sarcopenia is an age-related loss of muscle mass (atrophy of muscle fibers) and a decrease in specific force (weakness) (Doherty 2003). In humans, overt sarcopenia is present in 20% of 70 yr olds and 50% of 80 yr olds (Thomas 2010). Previously, we showed that sarcopenia affects the diaphragm muscle (DIAm) with a selective atrophy of type IIx and/or IIb muscle fibers and a reduction in maximum specific force (Elliott et al. 2016a; Gosselin et al. 1994; Greising et al. 2013, 2015b, 2015d). Functionally, we showed that DIAm sarcopenia impairs high-force motor behaviors necessary to clear the airways (Greising et al. 2015d). An inability to perform high-force motor behaviors required to clear the airways, such as sighing, coughing, and sneezing, may underlie respiratory complications that are particularly common in older humans (Enright et al. 1994; Polkey et al. 1997; Tolep et al. 1995).

Past studies of sarcopenia in limb muscles reported a loss of motor neurons (Brown 1972; Drey et al. 2014; Kwan 2013; Lexell et al. 1988; Tomlinson and Irving 1977), which would result in denervation of the muscle fibers comprising these motor units. Fiber type-specific effects of sarcopenia closely resemble those induced by DIAm denervation, particularly the atrophy of type IIx and IIb muscle fibers (Aravamudan et al. 2006; Argadine et al. 2009; Geiger et al. 2001, 2003; Gosselin et al. 1995; Mantilla et al. 2007; Miyata et al. 1995; Prakash et al. 1995, 1999; Sieck 1994; Sieck et al. 2007; Sieck and Zhan 2000; Zhan et al. 1995, 1997), leading us to hypothesize that sarcopenia is due to denervation resulting from an age-related loss of phrenic motor neurons (PhMNs). In this respect, we found that the DIAm contains different motor unit types (Fournier and Sieck 1988; Sieck et al. 1985; Sieck and Fournier 1987) and that type IIx and/or IIb muscle fibers comprise more fatigable fast motor units (Enad et al. 1989; Gransee et al. 2012; Sieck et al. 1989, 1996) that are recruited during high-force motor behaviors (Elliott et al. 2016b; Mantilla et al. 2010; Mantilla and Sieck 2011; Seven et al. 2014; Sieck 1991a, 1991b, 1994; Sieck and Fournier 1989).

Motor units are recruited systematically according to motor neuron size [Henneman size principle (Henneman et al. 1965)]. The size principle has been confirmed in the PhMN pool (Dick et al. 1987; Jodkowski et al. 1987; Mantilla et al. 2010; Seven et al. 2014; Sieck 1989, 1990, 1995, 1996; Sieck and Fournier 1989) and underlies DIAm motor unit recruitment to accomplish increasing force generation across a range of motor behaviors, with smaller PhMNs innervating type I and IIa DIAm fibers recruited first to accomplish low-force ventilatory behaviors and larger PhMNs innervating type IIx and/or IIb fibers recruited later to accomplish high-force airway clearance behaviors (Elliott et al. 2016b; Greising et al. 2012; Mantilla et al. 2010; Mantilla and Sieck 2011; Seven et al. 2014; Sieck 1988, 1989, 1990, 1995, 1996; Sieck et al. 1989; Sieck and Fournier 1989; Sieck and Prakash 1997). Thus we hypothesize that the selective effect of sarcopenia on type IIx and/or IIb fibers (Elliott et al. 2016a; Gosselin et al. 1994; Greising et al. 2013, 2015b, 2015d) and high-force motor behaviors (Greising et al. 2015c; Elliott et al. 2016a) reflects a selective loss of larger PhMNs.

METHODS

Animals.

Young (6 mo old, n = 10) and old (24 mo old, n = 10) female (n = 5 for both ages) and male (n = 5 for both ages) Fischer 344 pathogen-free rats were obtained from the National Institutes of Health (NIH) aged rodent colony, with design and age selection based on survival information (100% and 50%, respectively) (Miller and Nadon 2000). All protocols were approved by the Mayo Clinic Institute Animal Care and Use Committee (IACUC no. A57714) and complied with NIH and American Physiological Society guidelines (Drummond 2009).

Retrograde labeling of PhMNs.

PhMNs were retrogradely labeled with a procedure previously described (Mantilla et al. 2009; Obregon et al. 2009; Prakash et al. 1993, 1994, 2000; Sieck et al. 1999; Zhan et al. 1989). Briefly, after 48 h of acclimation, animals were anesthetized with ketamine (10 mg/kg) and xylazine (60 mg/kg), the left phrenic nerve was dissected in the neck by a ventral approach, and a 5-mm length of nerve was isolated and cut. The proximal phrenic nerve stump was then immersed in a dish containing 5 μl of 5% tetramethylrhodamine-conjugated dextrans (RhoD; Invitrogen, Carlsbad, CA) for 45–60 min before suturing. This method was chosen over labeling via intrapleural injection of cholera toxin B (Mantilla et al. 2009), as there is evidence of neuromuscular deficits in aging DIAm (Greising et al. 2015a, 2015b, 2015c, 2017; Lee et al. 2017; Prakash and Sieck 1998; Suzuki et al. 2009), which may affect complete labeling of the PhMN pool by intrapleural injection. The nerve dip technique bypasses the neuromuscular junction and effectively labels an entire unilateral PhMN pool, with no effect on the number of PhMNs (Mantilla et al. 2009).

Collection and processing of cervical spinal cord.

Three days after nerve dip, rats were anesthetized as above and then euthanized by transcardial exsanguination and perfused with heparinized saline before perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4).

The fixed cervical spinal cord was then excised via dorsal laminectomy and postfixed in 4% paraformaldehyde in PBS overnight and then immersed overnight in 25% sucrose in PBS before 50-μm longitudinal (horizontal) sections were cut with a cryostat (Frigocut; Reichert Microscope Services, Depew, NY). Sections were placed on gelatin-coated slides, treated with graded xylenes and ethanols, and coverslipped with DPX mounting media (Fluka, Sigma-Aldrich, St Louis, MO).

Confocal imaging of PhMNs.

Labeled PhMNs in the cervical spinal cord sections were visualized with an Olympus FV2000 laser confocal microscope (Olympus America, Melville, NY). A ×40 magnification objective (NA 1.4) was used to obtain a tissue mosaic visualization of the entire PhMN pool (Fig. 1). RhoD in the labeled PhMNs was excited with a 543-nm laser, and emission was detected at 625 nm. Images were captured in a 1,200 × 1,200 pixel array, with z stacks separated by 1.5 μm and similar acquisition parameters across preparations. Robust labeling of PhMNs was apparent in both ages studied (Fig. 1). Labeled PhMNs were counted from these z-stack mosaics in a manner identical to past reports (Alvarez-Argote et al. 2016; Mantilla et al. 2009; Prakash et al. 1993, 1994; Rana et al. 2017; Sieck et al. 1999).

Fig. 1.

Fig. 1.

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Robust labeling of PhMN pools in old and young rats. A and B: maximum-intensity z-stack projections of RhoD-labeled PhMN pools from young (A) and old (B) Fischer 344 rats. Scale bar, 100 μm. C: scatterplot (mean ± 95% CI) illustrating a decreased number of PhMNs in old rats (n = 10 rats per age). *P = 0.01, 2-way ANOVA with Bonferroni post hoc test.

Determination of PhMN size.

To determine PhMN size, we employed a method previously reported (Obregon et al. 2009; Prakash et al. 1993, 1994, 2000; Sieck et al. 1999). A systematic random sampling approach was used, in which every third PhMN from each rat (~55–80 PhMNs per animal) had the short- and long-axis lengths of the soma measured with the length tool in ImageJ (Schneider et al. 2012). In a subset of animals (6 mo n = 3 and 24 mo n = 3) the short- and long-axis somal lengths were measured for all PhMNs in the pool (~160–240 PhMNs, depending on age). PhMN somal surface areas were calculated with the formula for a prolate spheroid (Obregon et al. 2009; Prakash et al. 1993, 1994, 2000; Sieck et al. 1999; Ulfhake and Cullheim 1988). First-order dendritic segment lengths (somal edge to dendritic bifurcation) and mean diameters (at ~10 μm from the origin of the proximal segment) were measured with the line tool. An estimated surface area of the dendrites was calculated from the segment diameter with a previously validated quadratic model (Obregon et al. 2009). All measurements were performed at a midnuclear section (usually 3 or 4 per PhMN) within the z stack containing the entire PhMN, with overlapping of the somal boundaries avoided by judicious choice of an unambiguous z slice (Fig. 2). In cases where overlapping was unavoidable (<10% of PhMNs sampled), the next PhMN in the systematic random sampling regimen and an adjacent companion were assessed.

Fig. 2.

Fig. 2.

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Somal and dendritic morphometric measurements in RhoD-labeled PhMNs: representative midnuclear z slice from a RhoD-labeled PhMN. Somal morphometry was measured by circumscribing the PhMN soma (blue line) and measuring the short and long somal axes (red vectors). Dendritic surface area was estimated with validated formulas based on proximal dendritic diameters (yellow vectors). Scale bar, 25 μm.

Statistical methods.

The number of animals at each age was determined based on previously reported means and standard deviations of the number of PhMNs in rats (Alvarez-Argote et al. 2016; Mantilla et al. 2009; Rana et al. 2017) and an a priori power analysis (G*Power) to detect a physiologically relevant effect of ≥15% change in the number of PhMNs in old rats compared with young rats, assuming equal variances (Cohen’s d: 1.5). For comparisons of age and sex as factors affecting variance, two-way ANOVAs with Bonferroni post hoc tests were used. For comparisons of age, sex, and PhMN size as factors affecting variance, a three-way ANOVA with Bonferroni post hoc tests was used. For further analysis and graphical presentation, nonsignificant sex interactions were collapsed. All summary data sets used for this purpose exhibited normal distributions according to D’Agostino and Pearson normality tests. Differences in frequency distributions were assessed with Kolmogorov-Smirnov tests. Summary frequency distributions were assessed with nonparametric Kruskal-Wallis analysis and Dunn’s post hoc tests. Significance was set as P < 0.05, and all data are presented as means ± 95% confidence intervals (CIs) of the mean, unless otherwise stated.

RESULTS

Age-associated loss of PhMNs.

Only age had a significant effect on the number of PhMNs [F(1,16) = 24.3; P < 0.0002], with no difference between females and males [F(1,16) = 2.075; P = 0.17] or interaction between age and sex [F(1,16) = 0.21; P = 0.65] (Table 1, Fig. 1). By post hoc Bonferroni analysis, there were 22% fewer RhoD-labeled PhMNs in 24-mo-old rats compared with 6-mo-old rats (P = 0.01; Fig. 2). The mean numbers of PhMNs in females and males were within ~4% of each other in young rats and within ~10% of each other in old rats (Table 1).

Table 1.

PhMN number and summary somal morphometry of Fischer 344 rats

Property Young (n = 10) Old (n = 10) P Value
No. of PhMNs F: 229 ± 10 (n = 5) F: 174 ± 10 (n = 5) A: P = 0.0002*
M: 239 ± 9 (n = 5) M: 192 ± 12 (n = 5) S: P = 0.17
I: P = 0.65
Somal short-axis length, μm F: 24.4 ± 1.3 (n = 5) F: 20.3 ± 1.4 (n = 5) A: P = 0.002*
M: 23.5 ± 0.5 (n = 5) M: 19.0 ± 0.9 (n = 5) S: P = 0.34
I: P = 0.84
Somal long-axis length, μm F: 53.6 ± 1.5 (n = 5) F: 40.3 ± 2.2 (n = 5) A: P < 0.0001*
M: 58.6 ± 2.8 (n = 5) M: 39.7 ± 1.5 (n = 5) S: P = 0.31
I: P = 0.19
Somal surface area, μm2 F: 3,054 ± 154 (n = 5) F: 2,510 ± 1.5 (n = 5) A: P = 0.003*
M: 2,920 ± 170 (n = 5) M: 2,324 ± 167 (n = 5) S: P = 0.34
I: P = 0.88
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All data are means ± SE. Young rats are 6 mo old; old rats are 24 mo old. F, female; M, male; A, age; S, sex; I, interaction. All analyses are 2-way ANOVAs with Bonferroni post hoc test.

*

Significant difference. Post hoc significance is denoted by italicized values for old rats, where P < 0.05.

Age-associated changes in PhMN somal surface areas.

With z stacks of labeled PhMNs, morphometric properties of PhMNs in young and old Fischer 344 rats were determined (Table 1, Fig. 3). Only age had a significant effect on PhMN somal short-axis length [F(1,16) = 12.1; P = 0.003] and somal long-axis length [F(1,16) = 12.1; P = 0.003], with no difference between females and males or interaction between age and sex (Table 1). Post hoc Bonferroni analysis showed that the median PhMN somal short-axis length was 18% lower and the median PhMN somal long-axis length was 29% lower in old compared with young rats (P = 0.04 and P = 0.02, respectively; Table 1). Similarly, only age had a significant effect on PhMN somal surface area [F(1,16) = 12.1; P = 0.003], with no difference between females and males [F(1,16) = 0.96; P = 0.34] or interaction between age and sex [F(1,16) = 0.93; P = 0.88]. Post hoc Bonferroni analysis showed that the median PhMN somal surface area was 19% lower in old compared with young rats (P = 0.01; Fig. 3).

Fig. 3.

Fig. 3.

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Decreased PhMN somal surface area in old compared with young rats. A and B: representative high-magnification RhoD-labeled PhMN from a young (A) and an old (B) Fischer 344 rat. Scale bar, 25 μm. C: scatterplot (median ± 95% CI) of decreased PhMN somal surface area in old compared with young Fischer 344 rats. Summary data points are obtained from the median values of every third PhMN per rat (~55–80 PhMNs per sample). *P < 0.01, 2-way ANOVA with Bonferroni post hoc test. n = 10 rats per age. Red, females; blue, males.

Age-associated changes in distributions of PhMN somal surface areas.

The somal surface areas of PhMNs were calculated and plotted as frequency distributions (female: P < 0.0001, male: P < 0.0001, Komogorov-Smirnov; Fig. 4). The frequency distributions of PhMN somal surface areas in old rats were shifted leftward (P < 0.05, Kruskal-Wallis) compared with younger animals in both females (P = 0.002) and males (P < 0.0001), apparent from Dunn’s post hoc test in a cumulative frequency plot (Fig. 5). There was no difference between young females and young males (P > 0.99) or between old females and old males (P = 0.12).

Fig. 4.

Fig. 4.

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PhMN somal surface area of entire PhMN pool in young and old rats: frequency histograms of the number of PhMNs binned with respect to PhMN somal surface area in females (A) and males (B). Plotted is the entire PhMN pool from a single representative young (6 mo of age) and a single representative old (24 mo) Fischer 344 rat. K-S, Kolmogorov-Smirnov.

Fig. 5.

Fig. 5.

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Altered distribution of PhMN somal surface area in old compared with young rats: summary cumulative frequency distribution (mean ± 95% CI) illustrating a leftward shift (i.e., a preponderance toward smaller surface areas) in the PhMN somal surface areas of PhMNs from young (6 mo of age) and old (24 mo) female and male rats. Data were obtained from every third PhMN in each rat (~50–80 PhMNs per sample). Kruskal-Wallis (K-W) analysis with a Dunn’s post hoc test; age: P < 0.01; sex: P > 0.05. n = 10 rats per age.

Decreased number of larger PhMNs in old compared with young rats.

To further assess the reduction in the number of PhMNs in old rats, PhMNs were stratified into tertiles based on somal surface area values in young female and male rats. Age [F(1,2) = 8.4; P = 0.006], PhMN somal surface area tertile [F(2,2) = 14.0; P < 0.0001], and an interaction between tertile and age [F(2,2) = 12.2; P < 0.0001] had significant effects on the number of PhMNs. Sex had no effect on the number of PhMNs [F(1,2) = 0.005; P = 0.94], and the data were collapsed for post hoc tests. This analysis showed that there was a ~64% reduction in the number of PhMNs in the upper tertile of somal surface areas in old animals (P = 0.014; Fig. 6), while the number of PhMNs in the middle and lower tertiles of somal surface areas were unchanged.

Fig. 6.

Fig. 6.

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The loss of PhMNs during aging is almost exclusively of larger somal surface area neurons: scatterplot of the estimated number of PhMNs (mean ± 95% CI) within the phrenic motor pool in young (6 mo) and old (24 mo) Fischer 344 rats, stratified into lower, middle, and upper tertiles based on PhMN somal surface area at 6 mo. In old Fischer 344 rats, the relative prevalence of larger PhMNs is decreased by 64% compared with young Fischer 344 rats. *P = 0.014, 3-way ANOVA with Bonferroni post hoc tests, n = 10 rats per age, with sampling based on every third PhMN in the phrenic motor pool (~50–80 PhMNs per sample).

Age-associated changes in PhMN dendritic morphometry.

The mean number of proximal dendrites was unchanged in PhMNs between old and young rats, with no effect of age [F(1,16) = 1.3; P = 0.27], sex [F(1,16) = 0.4; P = 0.52], or interaction [F(1,16) = 0.2; P = 0.88; Table 2]. Similarly, the mean length of proximal dendrites in PhMNs was comparable between old and young rats, with no effect of age [F(1,16) = 0.3; P = 0.62], sex [F(1,16) = 0.8; P = 0.77], or interaction [F(1,16) = 1.1; P = 0.31; Table 2]. Only age had a significant effect on the total dendritic surface area [F(1,16) = 5.2; P = 0.04], with no difference between females and males [F(1,16) = 0.4; P = 0.84] or interaction between age and sex [F(1,16) = 0.2; P = 0.69]. Consolidating the sexes, the total dendritic surface area of PhMNs was 21% smaller in old compared with young rats (Table 2, Fig. 7A).

Table 2.

PhMN dendritic morphology in young and old Fischer 344 rats

Property Young (n = 10) Old (n = 10) P Value
No. of dendritic trees F: 5.6 ± 0.3 (n = 5) F: 5.2 ± 0.5 (n = 5) A: P = 0.27
M: 6.0 ± 0.3 (n = 5) M: 5.4 ± 0.7 (n = 5) S: P = 0.52
I: P = 0.88
Proximal segment length, μm F: 32.3 ± 4.6 (n = 5) F: 30.0 ± 3.0 (n = 5) A: P = 0.62
M: 26.7 ± 2.0 (n = 5) M: 33.2 ± 5.9 (n = 5) S: P = 0.76
I: P = 0.31
Estimated dendritic surface area, μm2 F: 6,419 ± 677 (n = 5) F: 5,310 ± 498 (n = 5) A: P = 0.04*
M: 6,782 ± 611 (n = 5) M: 5,192 ± 582 (n = 5) S: P = 0.84
I: P = 0.69
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All data are means ± SE. Young rats are 6 mo old; old rats are 24 mo old. F, female; M, male; A, age; S, sex; I, interaction. All analyses are 2-way ANOVAs with Bonferroni post hoc test.

*

Significant difference. Post hoc significance is denoted by italicized values for old rats, where P < 0.05.

Fig. 7.

Fig. 7.

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Decreased dendritic surface area of PhMNs in old compared with young rats. A: scatterplot (mean ± 95% CI) of reduced total dendritic surface area in old (24 mo) compared with young (6 mo) Fischer 344 rats (n = 10 rats per age). *P = 0.045, 2-way ANOVA—sexes consolidated. B: scatterplot of PhMN total dendritic surface area (mean ± 95% CI) within the phrenic motor pool in young (6 mo) and old (24 mo) Fischer 344 rats, stratified into lower, middle, and upper tertiles based on PhMN somal surface area at 6 mo. The dendritic surface areas of PhMNs from the upper tertile of young rats are larger than those of all lower and middle tertile PhMNs from both ages. Further analysis shows a 29% reduction in the estimated dendritic surface area of upper tertile PhMNs in old rats compared with young. *P = 0.003, 3-way ANOVA with Bonferroni post hoc tests, n = 10 rats per group.

Similar to the distribution of PhMN somal surface areas, there was a significant leftward shift in the distributions of total dendritic surface areas in old compared with young rats (Fig. 8). The frequency distributions of PhMN total dendritic surface areas in old rats were shifted leftward (P < 0.05, Kruskal-Wallis) compared with younger animals in females (P = 0.04) and males (P = 0.046), apparent from Dunn’s post hoc test in a cumulative frequency plot (Fig. 8). There was no difference between young females and young males (P > 0.92) or between old females and old males (P = 0.84).

Fig. 8.

Fig. 8.

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Altered distribution of PhMN dendritic surface area in old compared with young rats: summary cumulative frequency distribution (mean ± 95% CI) illustrating a leftward shift (i.e., a preponderance toward smaller surface areas) in the PhMN somal surface areas of PhMNs from young (6 mo) and old (24 mo) female and male rats. Data were obtained from every third PhMN in each rat (~50–80 PhMNs per sample). Kruskal-Wallis analysis with a Dunn’s post hoc test; age: P < 0.05; sex: P > 0.05; n = 10 rats per age.

To further assess the reduction in the mean dendritic surface area of PhMNs in old rats, we stratified PhMNs into lower, middle, and upper tertiles based on somal surface area values in young male and female rats (Fig. 7B). Age [F(1,2) = 8.3; P = 0.006], PhMN somal surface area tertile [F(2,2) = 9.5; P = 0.003], and an interaction between tertile and age [F(1,2) = 7.9; P = 0.04] had significant effects on the total dendritic surface area of PhMNs. Sex had no effect on the mean dendritic surface area of PhMNs [F(1,2) = 1.1; P = 0.31], and the data were collapsed for post hoc tests. As expected, the mean total dendritic surface area was larger in the young upper tertile of PhMN somal surface areas compared with the lower and middle tertiles at both ages (post hoc Bonferroni analysis; younglower vs. youngupper, P = 0.0005; youngmiddle vs. youngupper, P = 0.001; oldlower vs. youngupper, P < 0.0001; oldmiddle vs. youngupper, P < 0.0003). By contrast, the dendritic surface area of old upper tertile PhMNs was unchanged from lower and middle tertiles of all ages (younglower vs. oldupper, P > 0.99; youngmiddle vs. oldupper, P > 0.99; oldlower vs. oldupper, P > 0.99; oldmiddle vs. oldupper, P > 0.99). Furthermore, this analysis showed that there was an ~29% reduction in the total dendritic surface area of PhMNs in the upper tertile of somal surface areas in old animals (P = 0.004; Fig. 7B) compared with young rats. Within the lower and middle tertiles of somal surface areas, total dendritic surface areas of PhMNs were similar across ages (Fig. 7B).

DISCUSSION

The results of the present study demonstrated that in older Fischer 344 rats there are significantly fewer PhMNs (~22%) compared with young adult rats. This age-associated PhMN loss appears to predominantly affect the larger PhMNs, innervating type IIx and/or IIb DIAm fibers (Enad et al. 1989; Sieck et al. 1989, 1996). There was an age-associated shift in the distribution of somal PhMN morphological properties, such that surviving PhMNs in old Fischer 344 rats were smaller than in young rats. In addition, we observed a significant reduction in total dendritic surface area, particularly of the larger PhMNs in older Fischer 344 rats compared with young rats.

Taken together, these results support the concept that larger PhMNs are more vulnerable to age-related neural degeneration than smaller PhMNs. This loss of PhMNs may lead to the subsequent denervation of type IIx and/or IIb DIAm fibers and sarcopenia. The predominant age-related loss of larger PhMNs (a decline of ~64% compared with young rats) also explains the impairment of high-force airway clearance behaviors in older rodents (Greising et al. 2015d). The relative sparing of smaller PhMNs would also explain the resilience of lower-force ventilatory behaviors that require recruitment of only smaller fatigue-resistant motor units, which they innervate (Fournier and Sieck 1988; Mantilla et al. 2010; Mantilla and Sieck 2011; Seven et al. 2014; Sieck and Fournier 1989). Aging DIAm exhibits increased neuromuscular transmission failure (Greising et al. 2015b), with fast fatigue intermediate (FInt) and fast fatigable (FF) motor units known to be more susceptible (Ermilov et al. 2007; Mantilla et al. 2007; Rowley et al. 2007; Sieck and Prakash 1995), further underlining the differential vulnerability to aging across heterogeneous motor unit types.

Age-related motor neuron loss.

Previous reports suggest an age-related loss of motor neurons in the lumbar spinal cords of humans (Kawamura et al. 1985; Tomlinson and Irving 1977) and rodent models (Hashizume et al. 1988; Ishihara et al. 1987; Jacob 1998). One previous study also suggested an age-related loss of motor neurons in cervical spinal cord segments supplying the phrenic nerve, although PhMNs were not specifically identified (Zhang et al. 1996). In the present study, PhMNs were retrogradely labeled with a nerve dip technique that is very robust in labeling the entire PhMN pool in rats (Mantilla et al. 2009). Accordingly, the number of labeled PhMNs on the right side of young adult Fischer 344 rats (~234 PhMNs) was comparable to previous reports in adult (3 mo old) Sprague-Dawley rats (~230 PhMNs) (Alvarez-Argote et al. 2016; Mantilla et al. 2009; Prakash et al. 2000; Rana et al. 2017).

Motor units during aging.

A motor unit is the final motor output of the nervous system and consists of a motor neuron and all muscle fibers it innervates (Liddell and Sherrington 1925). Motor units are categorized into different types based on their contractile properties (slow vs. fast) and fatigue resistance [i.e., slow fatigue resistant (S), fast fatigue resistant (FR), FInt, and FF]. Smaller motor neurons comprise type S and FR motor units that include type I and IIa muscle fibers, respectively, which produce lower forces, while larger motor neurons comprise type FInt and FF motor units that include type IIx and/IIb fibers, which produce larger forces (Enad et al. 1989; Fournier and Sieck 1988; Sieck et al. 1989; Sieck and Fournier 1987). These different motor unit types are recruited according to motor neuron size [Henneman’s size principle (Henneman et al. 1965)], with smaller motor neurons of type S and FR motor units recruited first, followed progressively by larger motor neurons of type FInt and FF motor units (Elliott et al. 2016b; Mantilla et al. 2010; Mantilla and Sieck 2011; Seven et al. 2014; Sieck 1988, 1989, 1990, 1991b, 1995, 1996; Sieck et al. 1985; Sieck and Fournier 1989; Sieck and Prakash 1997). Orderly motor unit recruitment ensures smooth gradations of force to match functional demands, while avoiding fatigue for sustained low-force behaviors such as breathing.

Motor neuron loss and denervation.

In previous studies, we showed that unilateral denervation of the DIAm results in selective atrophy of type IIx and IIb fibers (Aravamudan et al. 2006; Argadine et al. 2009; Geiger et al. 2001; Gosselin et al. 1994; Miyata et al. 1995; Prakash et al. 1995, 1999; Sieck et al. 2007; Suzuki et al. 2009; Zhan et al. 1995) and a decrease in their specific force (Geiger et al. 2001). We also found that after unilateral DIAm denervation the generation of transdiaphragmatic pressures during ventilatory behaviors (eupnea and hypoxia/hypercapnia) was unaffected, whereas there was a reduction in transdiaphragmatic pressure generated during higher-force airway clearance behaviors (Gill et al. 2015; Khurram et al. 2017). Results of the present study showing a selective age-related loss of larger PhMNs are eminently consistent with the selective atrophy (sarcopenia) of type IIx and/or IIb DIAm fibers and the impairment of higher-force airway clearance behaviors with aging (Greising et al. 2015d).

Age-related changes in motor neuron morphology.

The present study shows an age-related decline in the total dendritic surface area of PhMNs, particularly of larger PhMNs. Reduced dendritic surface areas of PhMNs in older animals may disrupt the orderly recruitment of motor units (Torikai et al. 1996), causing a breakdown in regular neuro-motor control and further impairment of DIAm force generation (Mantilla et al. 2010, 2014a; Mantilla and Sieck 2011, 2013; Sieck 1991b, 1994; Sieck and Fournier 1989). The observation of an unchanged number of proximal dendrites of PhMNs is supported by similar observations in hypoglossal motor neurons in older Fischer 344 rats (Schwarz et al. 2009). Age-related loss of central synapses has been reported in rodent motor neurons (Matsumoto 1998), and age-related distal dendritic retraction is common in other neurons (Dickstein et al. 2007). However, the retrograde labeling technique we employed is able to fill only a portion of the dendritic tree, ~3rd to 4th order (Issa et al. 2010; Prakash et al. 2000). Future investigations using intracellular (Cameron et al. 1983, 1985; Kanjhan et al. 2016; Obregon et al. 2009) or Golgi (Goshgarian and Rafols 1981) approaches amenable to full morphometric and Sholl analysis (Klenowski et al. 2016, 2017) will provide useful information about aging-related changes of distal dendrites of PhMNs.

Mechanisms underlying age-related loss of larger PhMNs.

The precise mechanisms underlying age-related PhMN loss and reduction in size remain elusive. However, lessons from age-related neurodegenerative conditions that result in motor neuron death, such as amyotrophic lateral sclerosis (ALS), may prove useful in understanding the more protracted motor neuron loss during aging. In particular, ALS studies have also reported a loss of larger motor neurons (Kiernan and Hudson 1991). It has been suggested that this involves a disruption of trophic factor signaling including brain-derived neurotrophic factor (BDNF) and its high-affinity tropomyosin-related kinase B (TrkB) receptor, which promotes motor neuron survival in ALS models (Das et al. 2016; Henderson et al. 1994; Kishino et al. 1997). Thus a loss of trophic signaling seems to correlate with the loss of motor neurons in ALS. Similarly, the availability of TrkB receptors in PhMNs appears to decline during aging in the phrenic system (Greising et al. 2015b, 2017). In young adult rodents, acute inhibition of TrkB, repeated across a 7-day period, results in impaired neuromuscular transmission and reduced areas of neuromuscular junctions at type IIx and/or IIb fibers (Greising et al. 2015a; Mantilla et al. 2014b), mirroring deficits observed during old age (Greising et al. 2015b; Prakash and Sieck 1998; Valdez et al. 2012).

Mitochondrial dysfunction is strongly implicated in ALS, with fragmentation of mitochondria preceding neuronal death (Vande Velde et al. 2011; Vinsant et al. 2013). Strategies to inhibit mitochondrial fragmentation and enhance fusion appear to ameliorate motor neuron degeneration in ALS models (Song et al. 2013; Wang et al. 2013). Although controversial, TrkB receptors are shown to be present on the outer membrane of mitochondria (Wiedemann et al. 2006), which may underlie how neurotrophic signaling enhances mitochondrial fusion (Su et al. 2014). In any case, superoxide dismutase mutations, a cause of familial ALS and one of the most widely studied rodent models (Turner et al. 2013), have mitochondrial abnormalities and vacuolation (Fogarty et al. 2017; Kong and Xu 1998; Vande Velde et al. 2011; Vinsant et al. 2013) concomitant with functional changes in the entire neuraxis (Fogarty et al. 2015; Jiang et al. 2017; van Zundert et al. 2008) that precede motor neuron death. Taken together, an examination of age-related changes in PhMN mitochondria may provide useful insights into the mechanism of age-related motor neuron death.

Summary.

This study provides evidence for the age-related loss of predominantly the larger PhMNs in both female and male Fischer 344 rats and provides a foundation upon which to investigate the nuances of motor unit dysfunction during sarcopenia. It will be important to determine whether neural, neuromuscular junction, and skeletal muscle mechanisms of dysfunction are common or different across susceptible (S and FR) and vulnerable (FInt and FF) motor unit types.

GRANTS

This work was supported by National Institute on Aging Grant R01 AG-044615 (G. C. Sieck and C. B. Mantilla) and an Australian National Health and Medical Research Council CJ Martin Early Career Fellowship (M. J. Fogarty).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.J.F., T.S.O., C.B.M., and G.C.S. conceived and designed research; M.J.F., T.S.O., and W.-Z.Z. performed experiments; M.J.F. and G.C.S. analyzed data; M.J.F., C.B.M., and G.C.S. interpreted results of experiments; M.J.F. and G.C.S. prepared figures; M.J.F. and G.C.S. drafted manuscript; M.J.F., C.B.M., and G.C.S. edited and revised manuscript; M.J.F., T.S.O., W.-Z.Z., C.B.M., and G.C.S. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Yun-Hua Fang and Rebecca Macken for technical assistance in the completion of this project.

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