Mantilla, Carlos B.

ABSTRACT Motor neuron plasticity and survival depend on several trophic factors, with signaling via the tropomyosin receptor kinase B (TrkB) receptor being particularly important beyond development and across the lifespan. We hypothesized that differences in motor neuron properties may reflect differential trophic influences across motor units within a single motor unit pool. Based on extensive previous characterization of rat phrenic motor neurons (PhMN), somal surface area was used as an indicator of motor unit type (smaller neurons innervating slow‐twitch, fatigue‐resistant units versus larger neurons innervating fast‐twitch, fatigable units). To examine whether TrkB expression reflects these size‐related properties, TrkB mRNA transcripts were studied in individual PhMNs labeled retrogradely with Alexa Fluor 488‐conjugated cholera toxin subunit β from five adult male Sprague–Dawley rats. Longitudinal sections of the cervical C3–C5 spinal cord (10 μm thick) were processed using fluorescence in situ hybridization (RNAscope) and imaged using confocal microscopy to measure individual PhMN size and full‐length TrkB mRNA transcripts in nuclear and cytoplasmic compartments. Across 287 PhMNs, significant size variation existed both within and between animals. Across all animals, somal surface area averaged 4385 ± 1627 μm 2 with mean values per animal ranging from 3544 to 5461 μm 2 . PhMNs were classified into tertiles within each animal based on somal surface area. Individual PhMNs averaged 1070 ± 685 TrkB mRNA transcripts per motor neuron. Larger PhMNs in the upper tertile by size expressed a greater total number of TrkB mRNA transcripts per motor neuron, with values on average threefold higher compared to smaller motor neurons in the lower tertile ( p < 0.001). Cytoplasmic and nuclear TrkB mRNA density (transcripts/volume) displayed no significant effect of PhMN somal surface area tertile. Overall, the greater TrkB mRNA levels in larger motor units responsible for higher force, expulsive motor behaviors may underlie their functional adaptations and resilience to injury or disease. image

Neuromotor control of the diaphragm muscle (DIAm) after C 2 spinal hemisection (C 2 SH) is impacted differentially between ipsilateral and contralateral sides. We show neural drive to ipsilateral phrenic motor neurons decreases after C 2 SH, leading to a reduction in the number of DIAm motor units (MUs) recruited. Contralateral neural drive increases after C 2 SH, leading to an increase in the number of MU recruited. These results provide novel information about the impact of C 2 SH on DIAm neuromotor control.

Phrenic motor neurons (PhMNs) innervate the diaphragm muscle (DIAm), the primary pump muscle involved in breathing. It has been shown that brain-derived neurotrophic factor (BDNF) signals through its high-affinity, full-length tropomyosin receptor kinase B (TrkB.FL) to promote PhMN survival in embryonic and early postnatal development. Reduced BDNF/TrkB.FL signaling also appears to be involved in the age-related loss of PhMNs. Previously, we developed a novel chemogenetic rat model ( TrkB F616 rat) in which insertion of a knock-in allele sensitive to 1NMPP1 (a selective kinase inhibitor) allows rapid inhibition of TrkB.FL kinase activity. Using this TrkB F616 rat, we found that in younger animals, a 14-day period of 1NMPP1-induced TrkB.FL kinase inhibition resulted in an ~20% loss of larger PhMNs similar to that found with aging. Previous studies from our laboratory demonstrated that BDNF/TrkB.FL signaling induces pCREB S133 phosphorylation at serine residue 133 in PhMNs. 1NMPP1 induced inhibition of TrkB.FL kinase activity in TrkB F616 rats acutely blunts pCREB S133 phosphorylation induced by intrathecal BDNF treatment. In another cell type, we showed that pCREB S133 transcriptionally targets PGC1α expression with downstream upregulation of expression of mitochondrial transcription factor A (TFAM) and mitochondrial biogenesis. Based on a bioinformatic analysis, we identified a putative binding site for pCREB S133 in the rat PGC1α promoter. In the present study, we hypothesized that BDNF/TrkB.FL signaling mediates pCREB S133 phosphorylation and downstream activation of PGC1α thereby promoting mitochondrial biogenesis. Adult female and male TrkB F616 rats (~ 3 months of age; 300 g) were anesthetized (ketamine/xylazine) and either 25 μM 1NMPP1 or DMSO (vehicle control) was injected ip. After 2 h, the rats were treated intrathecally (infused at C 4 ) with 100 ng/ml BDNF for 40 min. Thereafter, the animals were euthanized and the ventral cervical spinal cord containing PhMNs was dissected, and lysates were prepared for subsequent analyses. Western blot was performed to quantify pCREB S133 phosphorylation, and PGC1α mRNA and protein expression were measured by qPCR and Western blot, respectively. A chromatin immunoprecipitation (ChIP) assay confirmed that pCREB S133 binds to the PGC1α promoter and that compared to vehicle treated controls, 1NMPP1induced TrkB.FL kinase inhibition reduced the binding affinity of pCREB S133 . Consistent with these results, we found that pCREB S133 phosphorylation and PGC1α mRNA and protein expression were reduced following 1NMPP1-induced TrkB.FL kinase inhibition. We conclude that in the rat cervical ventral horn, BDNF/TrkB.FL signaling induces pCREB S133 phosphorylation which transcriptionally activates PGC1α expression and downstream mitochondrial biogenesis. The BDNF/TrkB.FL signaling pathway acting in concert with activity dependent signaling may converge on pCREB S133 phosphorylation to maintain the survival of PhMNs by promoting mitochondrial biogenesis. The loss of the contribution of BDNF/TrkB.FL signaling in old age and with neurodegeneration may lead to PhMN death. Supported by NIH grants AG44615 and HL146114 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Neuromotor control of diaphragm muscle (DIAm) involves size-dependent recruitment of phrenic motor neurons (PhMNs). To accomplish breathing, only slow (type S) and fast, fatigue resistant (type FR) DIAm motor units are recruited. Thus, type s and FR DIAm motor units are very active in sustaining ventilation throughout life. Importantly, type S and FR DIAm motor units comprise small PhMNs that innervate type I and IIa DIAm fibers, respectively. More fatigable fast (type FF) motor units are very infrequently recruited for higher force, expulsive behaviors. Type FF DIAm motor units comprise large PhMNs that innervate type IIx/IIb DIAm fibers. Previously, we reported that compared to large PhMNs, small PhMNs have higher mitochondrial volume densities reflecting their more frequent activation. Using a quantitative histochemical technique to measure the maximum velocity of the succinate dehydrogenase reaction (SDH max ), we showed that type I and IIa DIAm fibers have higher oxidative capacity compared to type IIx/IIb fibers, which correlates with mitochondrial volume density and the more frequent activation of these muscle fibers. Accordingly, we hypothesize that the SDH max of small PhMNs is greater compared to large PhMNs. PhMNs in 6 month old, female and male Sprague Dawley rats were retrogradely identified by intrapleural injection of Alexa 647-conjugated cholera toxin B (CTB). Following retrograde CTB labeling, rats were perfused intracardially with a 0.1 M PBS, euthanized, and the C1–C6 segment of the spinal cord was dissected. Spinal cord sections were cut (40 mm) in longitudinal horizontal sections and mounted in 2-well Ibidi μ-slide plates. To measure SDH max , the sections were exposed to a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT; reaction indicator). As the SDH reaction proceeded, CTB-labeled PhMNs were imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion x60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD) was measured every 15 s over a 10-min period to measure the rate of accumulation of NBT diformazan (NBT dfz ; peak absorbance of 570 nm) in PhMNs. Using the Beer-Lambert equation, the change in OD was used to determine SDH max expressed as mmol fumarate/L cell/min. In separate experiments, following retrograde CTB labeling, mitochondria in PhMNs were labeled by transdural infusion of MitoTracker Red and imaged in 3D to determine mitochondrial volume density. Our results support a size-dependent heterogeneity in PhMN mitochondrial volume density and SDH max . We conclude that more active small PhMNs have a higher mitochondrial volume density and corresponding SDH max compared to large PhMNs. NIH grant AG44615 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Muscle plasticity is defined as the ability of a given muscle to alter its structural and functional properties in accordance with the environmental conditions imposed on it. As such, respiratory muscle is in a constant state of remodeling, and the basis of muscle's plasticity is its ability to change protein expression and resultant protein balance in response to varying environmental conditions. Here, we will describe the changes of respiratory muscle imposed by extrinsic changes in mechanical load, activity, and innervation. Although there is a large body of literature on the structural and functional plasticity of respiratory muscles, we are only beginning to understand the molecular‐scale protein changes that contribute to protein balance. We will give an overview of key mechanisms regulating protein synthesis and protein degradation, as well as the complex interactions between them. We suggest future application of a systems biology approach that would develop a mathematical model of protein balance and greatly improve treatments in a variety of clinical settings related to maintaining both muscle mass and optimal contractile function of respiratory muscles. © 2012 American Physiological Society. Compr Physiol 2:1441‐1462, 2012.

Abstract Striated respiratory muscles are necessary for lung ventilation and to maintain the patency of the upper airway. The basic structural and functional properties of respiratory muscles are similar to those of other striated muscles (both skeletal and cardiac). The sarcomere is the fundamental organizational unit of striated muscles and sarcomeric proteins underlie the passive and active mechanical properties of muscle fibers. In this respect, the functional categorization of different fiber types provides a conceptual framework to understand the physiological properties of respiratory muscles. Within the sarcomere, the interaction between the thick and thin filaments at the level of cross‐bridges provides the elementary unit of force generation and contraction. Key to an understanding of the unique functional differences across muscle fiber types are differences in cross‐bridge recruitment and cycling that relate to the expression of different myosin heavy chain isoforms in the thick filament. The active mechanical properties of muscle fibers are characterized by the relationship between myoplasmic Ca 2+ and cross‐bridge recruitment, force generation and sarcomere length (also cross‐bridge recruitment), external load and shortening velocity (cross‐bridge cycling rate), and cross‐bridge cycling rate and ATP consumption. Passive mechanical properties are also important reflecting viscoelastic elements within sarcomeres as well as the extracellular matrix. Conditions that affect respiratory muscle performance may have a range of underlying pathophysiological causes, but their manifestations will depend on their impact on these basic elemental structures. © 2013 American Physiological Society. Compr Physiol 3:1533‐1567, 2013.

Autophagy impairments have been implicated in various aging conditions. Previous studies in cervical motor neurons show an age-dependent increase in the key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. Chloroquine is commonly used to inhibit autophagy by preventing autophagosome–lysosome fusion and may thus emulate the effects of aging on the neuromuscular system. Indeed, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, consistent with aging effects. We hypothesized that chloroquine alters diaphragm muscle neuromuscular junction (NMJ) morphology and increases denervation. Adult male and female C57BL/6 × 129J mice between 5 and 8 months of age were used to examine diaphragm muscle NMJ morphology and denervation following daily intraperitoneal injections of chloroquine (10 mg/kg/d) or vehicle for 7 days. The motor end-plates and pre-synaptic terminals were fluorescently labeled with α-bungarotoxin and anti-synaptophysin, respectively. Confocal microscopy was used to assess pre- and post-synaptic morphology and denervation. At diaphragm NMJs, chloroquine treatment decreased pre-synaptic volume by 12% compared to the vehicle (p < 0.05), with no change in post-synaptic volume. Chloroquine treatment increased the proportion of partially denervated NMJs by 2.7-fold compared to vehicle treatment (p < 0.05). The morphological changes observed were similar to those previously reported in the diaphragm muscles of 18-month-old mice. These findings highlight the importance of autophagy in the maintenance of the structural properties at adult NMJs in vivo.

AbstractBackgroundSleep’s crucial role in maintaining brain health is increasingly recognized, particularly due to the rising prevalence of neurodegenerative diseases. It not only supports cognitive function but also aids in clearing brain metabolic waste through cerebrospinal fluid (CSF) dynamics. During sleep, especially in the non‐rapid eye movement (NREM) phase, CSF flow increases, essential for removing neurotoxic substances like amyloid‐beta proteins. This study explores the link between sleep respiratory patterns and CSF flow promotion.MethodElectroencephalography (EEG), and functional magnetic resonance imaging (fMRI) were simultaneously acquired during sleep from Gu, et al. (n = 29) (Gu et al. 2022). Respiration, measured via a respiratory belt, calculated respiratory volume (RV) as the standard deviation within a 5 second sliding window. EEG data processed delta band power (1‐4Hz) averaged across channels. Parcellated fMRI data using the brain template followed by the calculation of the averaged gray matter (GM) blood‐oxygen‐level‐dependent (BOLD) signal using the GM mask. CSF inflow was manually extracted from the fMRI’s bottom slice at the foramen magnum level. Subsequently, all signals were temporally aligned to the peak of the CSF inflow signal.ResultAt the time of CSF inflow (time = 0 sec. in the Figure 1A‐D), we observed an earlier increase in RV and EEG delta power, followed by changes in the GM BOLD signal (Figure 1). EEG and RV changes preceded the BOLD signal changes, which, in turn, preceded changes in CSF flow. Sensitivity to detect RV involvement during MRI varied with the placement of the respiratory belt, being least sensitive on the chest and more on the abdomen.ConclusionThese findings raise an interesting question on the role respiratory patterns during sleep, particularly in the NREM phase. Speculatively, RV change may influence CSF circulation during NREM sleep (Figure 1E). This enhancement in CSF flow is crucial for the clearance of metabolic waste from the brain, potentially reducing the risk of neurodegenerative diseases. Our findings indicate that respiratory volume may serve as a significant marker for CSF circulation during sleep.