Zhan, Wen‐Zhi

Neuroplastic changes in phrenic motor neurons that underlie recovery from spinal cord injury may be mediated by perineuronal nets. We show that changes in perineuronal nets around phrenic motor neurons occur within 7 days after cervical spinal cord injury, and that the increase in perineuronal nets around larger phrenic motor neurons was more pronounced. These changes are consistent with substantial reorganization of the neural network onto intact phrenic motor neurons following cervical spinal cord injury.

Brain-derived neurotrophic factor (BDNF) signals through its high-affinity full length tropomyosin receptor kinase-B (TrkB.FL). One key downstream target of BDNF/TrkB.FL signaling is phosphorylation of the transcription factor cyclic AMP response element-binding (CREB) protein at serine 133 (pCREBS133). We hypothesized that a positive feedback loop exists in the spinal cord in which BDNF/TrkB.FL induces pCREBS133 which then transcriptionally targets pro-BDNF expression. To test this hypothesis, we employed a loss of function approach using a novel chemogenetic rat model (TrkBF616A) with a knock-in allele sensitive to the selective kinase inhibitor 1NMPP1 to inhibit TrkB.FL kinase activity. In female and male TrkBF616A rats, we then examined the effects of 1NMPP1 (orally through drinking water) induced TrkB.FL kinase inhibition on BDNF levels in the cervical (CSC) and lumbar (LSC) spinal cord. The spinal cord was collected at two time points at 0700 and 010 reflecting the trough and peak timepoints of endogenous BDNF expression. BDNF levels were quantified using ELISA. We found in the CSC, BDNF levels did not vary across timepoints in either 1NMPP1 or vehicle treated groups. However, 1NMPP1-induced TrkB.FL inhibition significantly reduced BDNF levels at both timepoints (P=0.004). This reduction suggests that cervical circuits rely on continuous CREB-dependent support of BDNF expression and that inhibiting TrkB.FL disrupts this loop throughout the day. In the LSC, control rats displayed time of day-dependent variations in BDNF with lower levels at 0700 compared to 0100 (P=0.02). In contrast, 1NMPP1-induced inhibition of TrkB.FL reduced BDNF levels at 0100—when BDNF normally peaks in the LSC (P=0.01). This nighttime specific deficit supports the idea that activity-driven pCREBS133 activation is necessary to elevate BDNF during the active phase and that blocking TrkB.FL signaling disrupts this pCREBS133-mediated positive feedback. Overall, these results support a positive feedback that is region- and time-specific and involves BDNF/TrkB.FL signaling inducing pCREBS133 phosphorylation which then targets pro-BDNF expression in the spinal cord. Supported by NIH grants HL146114 and AG44615. This abstract was presented at the American Physiology Summit 2026 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.

Chloroquine is an antimalarial agent commonly used as an autophagy inhibitor by preventing autolysosomal fusion. Autophagy is an important cellular process through which defective cytoplasmic structures are recycled and degraded that has been implicated in aging neuromuscular effects. We recently showed that acute chloroquine treatment blunts the maximal forces generated by the diaphragm muscle, likely by impairing neuromuscular transmission generally consistent with aging effects in the diaphragm muscle. In addition, old mice show increased accumulation of autophagy proteins in phrenic motor neurons, consistent with impaired autophagy flux. In the present study, we hypothesized that chloroquine treatment results in accumulation of bulk endosomes pre-synaptically. Each hemidiaphragm with attached phrenic nerve was excised from 24-month-old male and female C57BL/6 mice to evaluate frequency dependent (0 vs 80 Hz stimulation) uptake of the styryl dye FM4-64, given that 80-Hz stimulation frequency induces bulk endocytosis after synaptic vesicle retrieval. Diaphragm-phrenic nerve preparations were incubated in 5 μM FM4-64 for 1 h prior to 6-min nerve stimulation (0.5 ms supramaximal pulses in 80 Hz, 200 ms trains delivered at 40% duty cycle) followed by a 5-min post-stimulation incubation. FM4-64 uptake at diaphragm neuromuscular junctions (NMJs) was measured using confocal microscopy. Preparations were randomly assigned to co-incubation with chloroquine (50 μM) or vehicle (Rees-Simpson solution). There was minimal FM4-64 uptake in the absence of phrenic nerve stimulation, consistent with the activity-dependent uptake of the styryl dye. With 80 Hz stimulation, high levels of FM4-64 uptake were evident in both the chloroquine and vehicle-treated groups without significant differences across groups. FM4-64 fluorescence was evident throughout the presynpatic terminal with few areas of dense accumulation, consistent with retention of endosomes. There was no evidence of greater endosome accumulation with chloroquine treatment. Of note, a slight increase in FM4-64 uptake was evident in the chloroquine treated groups compared to vehicle in the non-stimulated NMJs. These results are consistent with FM4-64 uptake reflecting bulk endocytosis that mainly occurs during high frequency stimulation. The impairment in autophagy flux following chloroquine treatment is expected to result in endosome accumulation but it is possible that the stimulation protocol was insuffcient to elucidate this possibility. Collectively, the findings of this study are consistent with impaired autophagy at diaphragm presynaptic terminals which may underlie neuromuscular deficits observed in old age. Supported by NIH grant R01 AG057052 and the Mayo Clinic. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In rats, there is a strong association between the emergence of mature diaphragm muscle (DIAm) myosin heavy chain isoform expression and the expansion of phrenic motor neuron (PhMN) size (from ~21-28 days postnatal (P)), alongside the manifestation of mature morphological and functional properties. In the Spastic ( Spa) mutant mouse, mutated glycine receptors lead to a spasticity phenotype emerging between P14-28, concomitant with DIAm and PhMN development. In adult Spa mice, DIAm weakness, fiber atrophy and loss of larger PhMNs is evident. We aim to characterize whether these DIAm and PhMN deficits occur contemporaneously to spasticity. Using wildtype (WT) and Spa mice at P14, P21, P28, and P90 (maturity) we evaluated DIAm and PhMN physiologic properties. Ex vivo DIAm strips were used for functional assessments of maximum specific force (Po) and fatigue. DIAm strips were also fresh-frozen, and 8 μm cross-sections were cut and stained with H&E to evaluate cross-sectional area (CSA) of individual DIAm fibers. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 μm) and prepared for confocal imaging and PhMN assessment. In WT mice, DIAm specific force increased at each age (P14, P21, P28 and P90), whilst in Spa mice, DIAm specific force was reduced from P21 compared to WT. Similarly, DIAm fiber CSA was reduced in Spa compared to WT mice at P21, P28 and P90. The number of PhMNs was similar between genotypes at P21, with a reduction in the number of PhMNs in Spa mice compared to WT evident at P28 continuing to P90. In Spa mice, PhMN size was smaller compared to WT at P21, unchanged at P28 and reduced at P90. The changes in Spa DIAm and PhMNs during the first postnatal month are consistent with the emergence of spasticity from P14 to P28. As most changes occur at ~P21, we are optimistic that interventions prior to weaning may promote the normal growth of DIAm and survival of PhMNs and may stave off later life DIAm behavioral deficits. HL166204 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

During early postnatal development, BDNF/TrkB signaling promotes PhMN survival. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact PhMN size. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact the number or CSA of DIAm fibers.

AbstractCervical spinal cord injury impacts ventilatory and non‐ventilatory functions of the diaphragm muscle (DIAm) and contributes to clinical morbidity and mortality in the afflicted population. Periodically, integrated brainstem neural circuit activity drives the DIAm to generate a markedly augmented effort or sigh—which plays an important role in preventing atelectasis and thus maintaining lung function. Across species, the general pattern of DIAm efforts during a normal sigh is variable in amplitude and the extent of post‐sigh “apnea” (i.e., the post‐sigh inter‐breath interval). This post‐sigh inter‐breath interval acts as a respiratory reset, following the interruption of regular respiratory rhythm by sigh. We examined the impact of upper cervical (C2) spinal cord hemisection (C2SH) on the transdiaphragmatic pressure (Pdi) generated during sighs and the post‐sigh respiratory reset in rats. Sighs were identified in Pdi traces by their characteristic biphasic pattern. We found that C2SH results in a reduction of Pdi during both eupnea and sighs, and a decrease in the immediate post‐sigh breath interval. These results are consistent with partial removal of descending excitatory synaptic inputs to phrenic motor neurons that results from C2SH. Following cervical spinal cord injury, a reduction in the amplitude of Pdi during sighs may compromise the maintenance of normal lung function.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

Neuromotor control of respiratory muscles such as the diaphragm muscle (DIAm), the main inspiratory muscle in mammals, is organized around motor units. Motor units comprise a motoneuron and the muscle fibers they innervate. Diversity in the contractile and fatigue properties of DIAm motor units permits generation of the wide range of behaviors necessary to sustain ventilation and maintain airways patent (e.g., sighs, which generate ~70% of maximal force in rats). Descending inputs to phrenic motoneurons innervating the DIAm is thought to be primarily unilateral in rats. After disrupting ipsilateral inputs by unilateral spinal cord hemisection at C2 (C2SH), latent, bilateral projections underlie recovery of ipsilateral DIAm activity. We hypothesized that DIAm EMG activity reflects bilateral descending premotor drive to motoneurons that varies across motor behaviors and conditions (anesthetized vs. awake). Using chronic DIAm EMG recordings in adult male Sprague‐Dawley rats, the bilateral vs. ipsilateral nature of premotor drive for each behavior and condition was evaluated by comparing the impact of C2SH. Three days prior to the C2SH surgery, a pair of DIAm electrodes was implanted bilaterally and DIAm EMG activity was assessed immediately prior to and after C2SH as well as at 3 days post‐C2SH in spontaneously‐breathing awake, restrained animals and following induction of ketamine‐xylazine anesthesia. DIAm EMG recordings comprised ~5 min of eupnea and exposure to hypoxia (10% O 2 )‐hypercapnia (5% CO 2 ). The completeness of C2SH surgery was confirmed by the absence of ipsilateral diaphragm EMG activity during eupnea at the time of C2SH surgery and at 3 days post‐C2SH in anesthetized animals, consistent with previous studies. In addition, histological assessment of disruption of anterolateral white matter tracts was conducted to verify the C2SH injury. Ipsilateral RMS EMG amplitude in anesthetized rats pre‐C2SH was ~30% and 45% of pre‐injury anesthetized sigh levels during eupnea and hypoxia‐hypercapnia, respectively. During awake conditions pre‐C2SH, ipsilateral RMS EMG amplitude was significantly higher (~50%, 70% and 200% of pre injury anesthetized sigh levels during eupnea, hypoxia‐hypercapnia and sighs, respectively). There were no differences across diaphragm muscle sides pre‐C2SH in RMS EMG amplitude. At 3 days post‐C2SH, ipsilateral RMS EMG activity was evident in anesthetized animals during hypoxia‐hypercapnia and sighs (~10% and 50% of pre‐injury anesthetized sigh levels) and in awake animals during eupnea, hypoxia‐hypercapnia and sighs (~25%, 50% and 125% of pre‐injury anesthetized sigh levels, respectively). These findings represent considerable differences in DIAm activity across motor behaviors and anesthetized vs. awake conditions and highlight physiologically‐relevant differences in the relative contribution of ipsilateral vs. bilateral inputs to phrenic motoneurons. Support or Funding Information NIH grant R01 HL96750.

During embryonic development, the number of phrenic motor neurons (PhMNs) declines precipitously during the final trimester and continues into the early postnatal period. When BDNF/TrkB signaling is absent in utero, animals do not survive after birth, most likely due to excessive loss of vital motor neurons (e.g., PhMNs). Far less is known about the early postnatal effect of BDNF/TrkB signaling. We have shown that BDNF/TrkB signaling is important in promoting PhMN neuroplasticity in mature rodents. Taken together, we hypothesized that inhibition of BDNF/TrkB signaling during early postnatal development results in fewer PhMNs. Chemo-genetic TrkBF616A mutant mice were used to induce inhibition of TrkB kinase activity in response to oral administration (via drinking water) of a phosphoprotein phosphatase 1 inhibitor derivative (1NMPP1). 1NMPP1-induced TrkB kinase inhibition was initiated at P0 and continued until P21 and compared to untreated control mice. At P28, PhMNs were retrogradely labeled via rhodamine phrenic nerve dip. One day later, mice were euthanized, perfused with 4% paraformaldehyde, and the cervical spinal cord was excised and processed for longitudinal cryosectioning (70 μm). Labeled PhMNs were imaged by 3D high resolution confocal microscopy. The number of PhMNs were counted. PhMN somal surface area and number of primary dendrites were also determined. 1NMPP1-induced inhibition of TrkB kinase resulted in 24% fewer PhMNs (P=0.01) and 21% more primary dendrites (P=0.0002) than control mice. The distribution of PhMN somal surface areas was not affected. We conclude that BDNF/TrkB signaling is essential in promoting survival of PhMNs in the early postnatal period. BDNF/TrkB signaling during postnatal development also plays a role in paring primary dendrites. While inhibition of TrkB kinase did not significantly alter the distribution of PhMN somal surface areas at P28, it is possible that further growth of PhMNs into maturity might have been altered as larger PhMNs emerge as a result of disproportionate growth beyond this age in mice. Supported by National Institutes of Health grants R01-AG044615 (GCS), R01-HL96750 (GCS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.