Citations

Excitatory descending glutamatergic (Glu) synaptic input to phrenic motor neurons (PhMNs) emanates from the ventrolateral medulla and is responsible for inspiratory drive and a range of higher force behaviors. Disruption of this excitatory input following unilateral C2 hemisection (C2SH) results in cessation of inspiratory‐related diaphragm muscle (DIAm) activity ipsilateral to injury, but DIAm activity during higher force behaviors persists, reflecting the contribution of spared contralateral inputs. In a model of PhMN (DIAm motor unit) recruitment, we proposed that smaller PhMNs innervating more fatigue resistant DIAm motor units are recruited to accomplish ventilatory behaviors, whereas larger PhMNs innervating more fatigable motor units are recruited to accomplish more forceful expulsive and straining behaviors. We hypothesized that differences in the distribution of ipsilateral and contralateral Glu synaptic inputs to PhMNs contribute to the effects of C2SH on the range of motor behaviors accomplished by the DIAm. Accordingly, we hypothesized that C2SH primarily disrupts ipsilateral Glu synaptic inputs to smaller PhMNs, whereas Glu synaptic inputs to larger PhMNs are minimally impacted after C2SH. To address this hypothesis, we examined changes in Glu presynaptic input onto retrogradely labeled PhMNs using immunohistochemistry for VGLUT1 and VGLUT2. We found that following C2SH (by 7 days), there was a ~45% reduction in ipsilateral Glu synaptic input to PhMNs compared to a ~20% reduction contralaterally. In addition, ipsilateral to C2SH, Glu synaptic input to smaller PhMNs was reduced to a greater extent compared to that to larger PhMNs (55 vs. 35% reduction, respectively). Contralateral to C2SH, inputs to PhMNs were similarly reduced independent of motor neuron size. These results are consistent with more uniformly distributed contralateral inputs to all PhMNs and predominant input to smaller PhMNs ipsilaterally. These results also indicate that the source of Glu synaptic input to PhMNs varies depending on motor neuron size and likely reflects different motor control, including possibly distinct central pattern generator and premotor circuits. Support or Funding Information Grant Support: NIH Grant R01 HL96750 and Mayo Clinic Graduate School of Biomedical Sciences This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Brain derived neurotrophic factor (BDNF) acting through the high affinity tropomyosin‐related kinase receptor B (TrkB) plays an important role in synaptic plasticity and function at the neuromuscular junction (NMJ) as well as maintenance of neuromuscular transmission. Our previous studies showed that susceptibility to age‐related changes in diaphragm muscle (DIAm) NMJ morphology and denervation depends on trophic influences exerted by BDNF/TrkB signaling. Indeed, TrkB kinase activity is necessary to stabilize DIAm NMJ pre‐synaptic terminals in 18 month old mice (representing early old age; ~90% survival). However, by 24 months of age (~75% survival), BDNF/TrkB signaling has no effect on DIAm neuromuscular transmission, suggesting reduced TrkB kinase activity in the older age groups. We hypothesized that the NMJs that are resilient to old age do not depend on TrkB kinase activity for stabilization of pre‐synaptic terminals. Male and female 24 month old TrkB F616A mice were used to examine the structural properties of NMJs across various muscles with varying susceptibility to age‐related effects (DIAm, EDLm, and SOLm). Confocal microscopy was used to compare pre‐ and post‐synaptic morphology and denervation following a 7 day treatment with the phosphoprotein phosphatase‐1 derivative 1NMPP1, which inhibits TrkB kinase activity in TrkB F616A mice vs. vehicle treatment. Inhibition of TrkB kinase activity had no effect on pre‐synaptic or post‐synaptic terminal volume in DIAm, EDLm, and SOLm. Similarly, the overlap of pre‐ and post‐synaptic structures and the proportion of denervated NMJs did not change with 1NMPP1 treatment in all muscle types. The average percent of denervated NMJs was 8, 22, and 7% in DIAm, EDLm, and SOLm, respectively. Collectively these results suggest that the requirement of TrkB kinase activity for stabilization of NMJs in DIAm, EDLm and SOLm wanes in old age. Support or Funding Information Supported by R01‐AG057052. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

In skeletal muscles, force gradation depends on the level of motor unit (MU) activation. MU recruitment generally follows a specific order, with MUs comprising type I or type IIa fibers more active than type IIx and/or IIb. Direct evidence of MU recruitment across motor behaviors is limited to samples of relatively few MUs within a pool. The purpose of this study was to determine the extent of diaphragm MU recruitment across different motor behaviors. We hypothesized that in vivo labeling of individual neuromuscular junctions (NMJs) by FM4‐64 reflects the activation of MUs . Adult rats were exposed to eupneic (E), hypoxic/hypercarbic (H) conditions or supramaximal phrenic nerve stimulation (S). Individual diaphragm NMJs were type‐identified by myosin heavy chain expression. Average FM4‐64 labeling for NMJs at type I or IIa fibers vs. type IIx and/or IIb fibers increased progressively from E to H to S. The proportion of labeled NMJs of each type also increased significantly. At type I or IIa fibers, 40%, 59% and 90% of NMJs were labeled during E, H and S, respectively. At type IIx and/or IIb fibers, 21%, 32% and 82% of NMJs were labeled during E, H and S, respectively. Thus, using an in vivo labeling method there is direct evidence of progressive MU recruitment across different ventilatory and non‐ventilatory behaviors in the rat diaphragm. Supported by NIH grants HL37680 and AR51173, and the Mayo Clinic.

TAR DNA‐binding protein of 43 kDa (TDP‐43) is a nuclear protein involved in transcriptional repression and alternative splicing. It has been shown to be a component of ubiquitin‐positive, tau‐ and á‐synuclein‐negative inclusions in neuronal soma and nuclei as well as neurites of frontotemporal lobar degeneration (FTLD‐U) and in motor neurons of amyotrophic lateral sclerosis (ALS). Recently, it has been co‐localized with Lewy bodies in Lewy body disease (LBD). In this study we use immunogold electron microscopy to study the ultrastructural features of TDP‐43‐labeled structures. The results showed that in all FTLD‐U, ALS and LBD, TDP‐43 was localized to filaments coated with granular material. Both coated and uncoated regions of the filaments were labeled. In FTLD‐U a subset of neurites filled with densely packed filaments were also labeled. The filaments have a diameter of 10–17 nm. Neurofilament and glial filaments were not labeled by TDP‐43. The data suggests that TDP‐43 is a constituent protein in filamentous inclusions in neurodegenerative diseases. Supported by NIH grant AG17216.

Retrograde transport of fluorescent dyes permits imaging of nearly the entire pool of motoneurons when administered in the target muscle or axon shaft, but the extent of dendritic labeling is usually limited. We used an intracellular injection of Lucifer Yellow CH (LY) into retrogradely labeled phrenic motoneurons (PMNs) to enhance dendritic structure. PMNs were specifically labeled by Alexa Fluor 488‐conjugated cholera toxin fragment B (CTB) injected into the pleural space of 18‐days old rats. After a 72‐hr survival period, animals were transcardially perfused with 4% paraformaldehyde and 4% sucrose in 0.1 M phosphate buffer. Cervical spinal cords were sectioned in coronal plane. Individual CTB‐labeled PMNs were intracellularly injected with LY under direct visual control and subsequently imaged with an Olympus FluoView 200 laser‐scanning confocal system. Image processing and analyses were performed using Metamorph 7.4 software. PMNs were extensively labeled and reconstructed in 3D. The range of visible primary, secondary and tertiary processes was 3 ‐ 7, 2 ‐ 4 and 0 ‐ 2 per PMN, respectively. Average length of a single dendritic tree was 218.0±20.1 μm and surface area was 1295±199 μm 2 . The described technique provides 3D quantitative morphometry of a large sample of retrogradely‐labeled PMNs in great detail. Supported by NIH grants HL37680 and AR51173.

Unilateral denervation (DNV) of rat diaphragm muscle (DIAm) increases protein synthesis and degradation, with net protein breakdown by 14 days after DNV. We hypothesized that neural influences such as the nerve‐derived trophic factor neuregulin (NRG‐1) are essential in maintaining DIAm protein balance. NRG activates receptor tyrosine kinases of the ErbB family, with the ErbB2‐ErbB3 co‐receptor showing highest affinity for NRG‐1 and strong phosphoinositol‐3‐kinase (PI3K) activation. Both protein synthesis (via Akt phosphorylation) and degradation (via the transcription factor FoxO3A) are regulated by PI3K activity. We used Western blot analyses to assess NRG‐1, ErbB2, ErbB3 and FoxO3A protein expression at 1, 3, 7, and 14 days after DNV. By 1 day after DNV, NRG‐1 expression decreased, whereas ErbB2 and ErbB3 expression increased. Consistent with reduced NRG/ErbB signaling, association of the PI3K regulatory subunit p85 with ErbB3 decreased at 14 days after DNV. FoxO3A expression increased at 7 and 14 days after DNV, in agreement with delayed net protein breakdown. Importantly, DIAm inactivity induced by spinal hemisection had no effect on ErbB2, ErbB3, or FoxO3A expression. Taken together, these results indicate that DNV‐induced skeletal muscle adaptations result from removal of a trophic influence such as NRG‐1 rather than inactivity itself. Supported by NIH grant AR51173.

Muscle protein content reflects a balance between protein synthesis and degradation, which are likely regulated by neuromuscular activity and/or neurotrophin influence. In previous studies, we found that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) protein content after approximately 7 days. However, we did not determine whether the decrease in DIAm MHC protein content was the result of a decrease in protein synthesis, an increase in protein degradation, or both. We hypothesize that protein degradation plays a significant role in the decrease in DIAm protein content following DNV. In this study, we analyzed the ubiquitination state of cellular proteins as a marker of protein degradation. Conjugation of ubiquitin to cellular proteins targets proteins for degradation by proteosomes. We analyzed DIAm from adult male Sprague‐Dawley rats 3, 7 and 14 days after DNV. The DIAm was homogenized and protein ubiquitination was analyzed by Western analysis. No difference in ubiquitination was detected between control (CTL), sham surgery‐treated (sham), and 3‐day DNV animals. However, following 7 and 14 days of DNV, there was an approximate five‐fold increase in protein ubiquitination in DIAm compared to CTL and sham groups. These results demonstrate that DIAm protein ubiquitination increases between 3 and 7 days following DNV, and support the hypothesis that protein degradation contributes to the DNV‐induced decrease in DIAm MHC protein content. Supported by NIH grants AR51173 and HL37680.

Following spinal cord hemisection at C2 (SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle paralysis with partial spontaneous recovery of rhythmic phrenic activity over time. Brain‐derived neurotrophic factor (BDNF) plays an important role in neuroplasticity following SH. Intraspinal transplantation of mesenchymal stem cells engineered to produce BDNF‐GFP (BDNF‐MSCs) at the time of injury promotes functional recovery of all animals, despite low levels of MSC survival at 14 days post‐SH. The goal of the present study was to determine the survival and localization of BDNF‐MSCs after intraspinal transplantation. BDNF‐MSCs were injected intraspinally at C4 (2 sites, total 2 x 10 5 cells in 4 µl) in adult male Sprague‐Dawley rats. After one and three days, the cervical spinal cord was fixed, excised, and cut longitudinally. Survival of transplanted BDNF‐MSCs was confirmed in all animals at 1 day post‐transplantation (n=5) and in five of 6 animals at 3 days post‐transplantation. BDNF‐MSCs remained localized at the site of injection in the white matter at 1 and 3 days, with an average volume of 1.1 ± 0.3 µl. There was no localization of MSCs in the vicinity of phrenic motoneurons identified by retrograde labeling with cholera toxin subunit B. Understanding the time course of stem cell survival after transplantation is important in defining windows of opportunity during which the beneficial effects of BDNF on respiratory function after spinal cord injury are executed. Supported by HL96750 and Mayo Clinic Center for Regenerative Medicine.