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.

Spinal cord hemisection at C2 (SH) disrupts descending drive to ipsilateral phrenic motoneurons (PMNs) causing unilateral diaphragm muscle (DIAm) paralysis. Previously, we found a decrease in motoneuron size at 2 weeks post‐SH. Recently, we found neuregulin (NRG) in synaptic boutons surrounding PMNs. NRG is a trophic factor known to regulate development of the neuromuscular junction, but the effect of NRG on motoneurons is unknown. We hypothesized that 1) NRG input to PMNs is supraspinal in origin, and 2) SH disrupts NRG input to PMNs. Adult male Sprague‐Dawley rats underwent SH or sham‐surgery. PMNs were retrogradely labeled by intramuscular injection of Alexa488‐conjugated cholera toxin B. Using laser capture microdissection, we found that PMNs express mRNA for the NRG receptors ErbB2 and ErbB3, providing a functional substrate for a NRG effect. Two weeks post‐SH, labeled PMNs and NRG boutons were imaged using confocal microscopy. PMN morphology and the number of NRG boutons in the vicinity of PMNs were determined using a computerized, semi‐automated reconstruction algorithm. NRG boutons were fairly homogeneously distributed on motoneurons independent of size. Following SH, few NRG boutons were observed. We conclude that 1) NRG input to PMNs derives from a supraspinal source; and 2) SH removes NRG input to PMNs. It is possible that NRG input exerts a trophic influence on PMNs. Supported by NIH grants HL37680, AR51173 and Mayo Clinic

Cardiovascular reactivity to stress may be predictive of cardiovascular disease. Sympathoexcitatory responses to mental stress (MS), cold pressor test (CPT), isometric handgrip (HG) and head‐up tilt (HUT) target different physiological mechanisms, but the correlation between response variables is unclear. Our purpose was to test the hypothesis that pressor and catecholamine responses between these stressors would be correlated within subjects. Healthy, normotensive young adults (n=76) underwent MS, CPT, HUT and HG to fatigue. Arterial pressure and HR were monitored, and arterial Epi and NE were sampled during the final 30 sec of each stressor. Percent change from baseline was averaged during MS, CPT, HUT and the final 20% time segment of HG. MAP and SBP were positively correlated between CPT and HG (r = 0.46, 0.52, resp, p < 0.001) but not among other tests. Interestingly, the % increase in NE response to all 4 maneuvers showed robust correlations (MS v CPT r = 0.65; MS v HG r = 0.74; MS v HUT r = 0.53; CPT v HG r = 0.72; CPT v HUT r = 0.68; HG v HUT r = 0.59; p < 0.001 for all). Similar correlations were present for Epi except for comparisons with HUT. This suggests that among the 4 tests, HR and BP responses are generally variable, but NE and Epi responses are remarkably consistent (except Epi for HUT), implying that lab stressors may evoke sympathoexcitation more similarly than previously thought. Support: K23‐RR17520, HL 46493.

Neuregulin (NRG) is a nerve derived trophic factor that regulates acetylcholine receptor expression during neuromuscular junction development. NRG also has important trophic effects in the adult central nervous system. We hypothesized that NRG is a trophic factor for phrenic motoneurons. In adult male Sprague‐Dawley rats, we examined NRG‐1 immunoreactivity in the vicinity of phrenic motoneurons. Retrogradely‐labeled phrenic motoneurons were imaged and reconstructed in 3D. NRG‐1 boutons were found mainly around the soma and primary dendrites. The co‐localization of NRG boutons and synaptic markers was determined using multicolor confocal microscopy and a custom‐developed application (Metamorph; Universal Imaging). We found that 94 ± 3% of NRG‐containing boutons resided on synapses, as determined by co‐localization with synaptophysin. In addition, 6 ± 3% of synapses on phrenic motoneurons (within 1 micrometer of soma and primary dendrite surface) expressed NRG. Overall, 13 ± 5 NRG boutons were present at the soma vs. 6 ± 3 at each primary dendrite of a motoneuron.Thus, 57% of NRG‐containing boutons were surrounding the motoneuron soma. In summary, NRG‐1 synaptic boutons are present at phrenic motoneurons in the adult, although its role is presently unknown. Supported by NIH grant AR51173.

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.