Aging affects the number and morphological heterogeneity of rat phrenic motor neurons and phrenic motor axons

Abstract

Diaphragm muscle (DIAm) motor units comprise a phrenic motor neuron (PhMN), the phrenic nerve and the muscle fibers innervated, with the size of PhMNs and axons characteristic of motor unit type. Smaller PhMNs and their axons comprise slow (type S) and fatigue‐resistant (type FR) DIAm motor units, while larger PhMNs and their axons comprise more fatigable (type FF) motor units. With aging, we have shown a loss of larger PhMNs, consistent with selective atrophy of type IIx/IIb DIAm fibers and reduced maximum DIAm force. In the present study, we hypothesized that with aging there is a loss of larger myelinated phrenic α motor axons. Female and male young (6 months) and old (24 months) Fischer 344 rats were studied. PhMNs were retrogradely labeled by intrapleural injection of 488‐conjugated CTB. The phrenic nerves were excised ~1 cm from the DIAm insertion and mounted in resin, and phrenic α motor axons were delineated based on size (i.e., >4 μm diameters). In older rats, the number of larger PhMNs and larger phrenic α motor axons were reduced. There were no differences in non‐α axons. In addition, there was evidence of demyelination of larger phrenic α motor axons in older rats. Together, these findings are consistent with the selective age‐related vulnerability of larger PhMNs and denervation of type FF motor units, which may underlie DIAm sarcopenia.

Dendritic morphology of motor neurons and interneurons within the compact, semicompact, and loose formations of the rat nucleus ambiguus

“…Overall surface areas of nucleus ambiguus MNs of Fischer 344 rats were slightly smaller than other brainstem (i.e., hypoglossal) and spinal cord (i.e., phrenic) MNs in Fischer 344 rats (Fogarty and Sieck, 2023). Although larger MNs are more likely to comprise fast fatiguable motor units compared to smaller MNs, comprising slow or fast fatiguable MNs (Burke et al, 1973;Dick et al, 1987;Heckman and Enoka, 2012;Fogarty and Sieck, 2019).…”

“…The larger range of dendritic sizes is consistent with a more diverse motor unit population, confirmed by the mixed myosin heavy chain expressions confirming type I, IIa, IIx, IIb, and mixtures of IIx and IIb with extraocular fibres in cricothyroid, cricoarytenoid, and thyroarytenoid muscles ( Rhee et al, 2004 ). Thus, within the semicompact/loose formations of the nucleus ambiguus there resides age- and neurodegeneration-vulnerable and resilient MNs innervating the larynx, similar to other mixed motor unit population motor pools ( Fogarty et al, 2018 ; Fogarty, 2023 ; Fogarty and Sieck, 2023 ). This is borne out in observations of dysphagia and dysphonia in the elderly, in Alzheimer’s disease and amyotrophic lateral sclerosis sufferers ( Turley and Cohen, 2009 ; Humbert et al, 2010 ; Christmas and Rogus-Pulia, 2019 ; Garand et al, 2022 ), alongside rodent studies of aged Fischer 344–Brown Norway crosses ( Basken et al, 2012 ; Krekeler et al, 2020 ).…”

“…Indeed, despite being smaller than hypoglossal MNs, laryngeal, pharyngeal, and oesophageal muscles express palpable levels of the myosin heavy chain type IIx (MyHC 2X ) ( Rhee et al, 2004 ) or lower succinate dehydrogenase and mitochondrial volume density ( Hinrichsen and Dulhunty, 1982 ), consistent with being fast intermediate (fatiguable) motor units ( Brown et al, 2021b ). Based on the past findings in aged Fischer 344 rats ( Hashizume et al, 1988 ; Jacob, 1998 ; Kanda and Hashizume, 1998 ; Fogarty et al, 2018 ; Fogarty, 2023 ; Fogarty and Sieck, 2023 ) and facial ( Johnson and Duberley, 1998 ) and findings in Fisher–Brown Norway crosses ( Basken et al, 2012 ), we would expect nucleus ambiguus MNs to be vulnerable to age-associated death. This vulnerability would be consistent with age-associated dysphonias and dysphagias ( Turley and Cohen, 2009 ; Basken et al, 2012 ; Marino and Johns, 2014 ; Thiyagalingam et al, 2021 ).…”

Myelinated peripheral axons are more vulnerable to mechanical trauma in a model of enlarged axonal diameters

“…Considering that (1) axons display a shift toward smaller diameters in our mild nerve compression model, (2) Cmtm6 ‐cKo mice before injury display a shift toward larger axonal diameters and (3) early post‐injury present with even smaller axonal diameters and an increased number of pathological profiles that (4) coincide with an enhanced immune response, we believe that the enlargement of the diameters of peripheral axons in Cmtm6 ‐cKo mice enhances the vulnerability to mechanical impact. The concept that axons of larger diameters are more susceptible to becoming pathological and to degenerate after injury has been previously suggested based on observations in demyelinating/neurodegenerative disorders in both PNS and CNS (Fujimura et al, 1991; Li, 2015; Lovas et al, 2000; Vavlitou et al, 2010), traumatic brain injuries (Alexandris et al, 2022; Mierzwa et al, 2014), nerve compression injuries (Strain & Olson, 1975), and during aging in the PNS (Chase et al, 1992; Fogarty & Sieck, 2023). Our data are also in agreement with a recent report that a partial nerve crush injury spares small‐diameter axons from degenerating and preserves afferent fiber innervation with the skin.…”