Phrenic Motoneurons Research Articles

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia

Plasticity is an important aspect of the neural control of breathing. One well-studied form of respiratory plasticity is phrenic long-term facilitation (pLTF) induced by acute intermittent but not sustained hypoxia. Okadaic acid-sensitive protein phosphatases (PPs) differentially regulate phrenic nerve activity with intermittent vs. sustained hypoxia, at least partially accounting for pLTF pattern sensitivity. However, okadaic acid inhibits multiple serine/threonine phosphatases, and the relevant phosphatase (PP1, PP2A, PP5) for pLTF pattern sensitivity has not been identified. Here, we demonstrate that sustained hypoxia (25 min, 9-10.5% O2) elicits phrenic motor facilitation in rats pretreated with bilateral intrapleural injections of small interfering RNAs (siRNAs; Accell-modified to preferentially transfect neurons, 3.33 μM, 3 days) targeting PP1 mRNA (48 ± 14% change from baseline, n = 6) but not PP2A (14 ± 9% baseline, n = 6) or nontargeting siRNAs (4 ± 10% baseline, n = 7). In time control rats (no hypoxia) treated with siRNAs ( n = 6), no facilitation was evident (-9 ± 9% baseline). siRNAs had no effect on the hypoxic phrenic response. Immunohistochemistry revealed PP1 and PP2A protein in identified phrenic motoneurons. Although PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, we were not able to verify "knockdown" in vivo after siRNA treatment. On the other hand, PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, verifying the intended siRNA effects. In conclusion, PP1 (not PP2A) is the relevant okadaic acid-sensitive phosphatase constraining phrenic motor facilitation after sustained hypoxia and likely contributing to pLTF pattern sensitivity. NEW & NOTEWORTHY This study demonstrates that the relevant okadaic acid-sensitive Ser/Thr protein phosphatase (PP) constraining facilitation after sustained hypoxia is PP1 and not PP2A. It suggests that PP1 may be critical in the pattern sensitivity of hypoxia-induced phrenic motor plasticity.

Microsurgical technique for concurrent cervical corpectomy and ventral phrenic nerve access in the rat

The phrenic nerve is useful to record as a motor output in studies investigating neural control of respiration. It may be accessed via dorsal or ventral microsurgical approaches. Since such studies frequently involve concurrent access to the spinal cord, the two approaches may be alternatively used, each with its own set of advantages and disadvantages. The dorsal approach permits easier exposure of the spinal cord via laminectomy, but, compared to the ventral approach, phrenic nerve access proves more challenging, and concurrent surgical access to the full complement of respiratory-related nerves (i.e., glossopharyngeal, vagus, recurrent laryngeal, hypoglossal nerves) and cervical sympathetic nerve in the neck is limited. The ventral approach achieves more direct access to the phrenic and respiratory-related nerves, but ventral access to the spinal cord via corpectomy requires much greater diligence and vigilance. Ventral spinal cord access, however, facilitates neuronal (e.g., phrenic motoneuron and interneuron) recordings in the ventral horn of the spinal cord, given greater proximity to the ventral compared to the dorsal surface of the spinal cord providing more leeway in recording pipette insertion point and trajectory. Additionally, ventral access to the cervical spinal cord proves useful across a broad range of studies investigating normal spinal cord physiology as well as spinal cord injury. We detail the microsurgical technique of concurrent ventral phrenic nerve dissection and cervical corpectomy in adult rats.

Local BDNF Delivery to the Injured Cervical Spinal Cord using an Engineered Hydrogel Enhances Diaphragmatic Respiratory Function.

We developed an innovative biomaterial-based approach to repair the critical neural circuitry that controls diaphragm activation by locally delivering brain-derived neurotrophic factor (BDNF) to injured cervical spinal cord. BDNF can be used to restore respiratory function via a number of potential repair mechanisms; however, widespread BDNF biodistribution resulting from delivery methods such as systemic injection or lumbar puncture can lead to inefficient drug delivery and adverse side effects. As a viable alternative, we developed a novel hydrogel-based system loaded with polysaccharide-BDNF particles self-assembled by electrostatic interactions that can be safely implanted in the intrathecal space for achieving local BDNF delivery with controlled dosing and duration. Implantation of BDNF hydrogel after C4/C5 contusion-type spinal cord injury (SCI) in female rats robustly preserved diaphragm function, as assessed by in vivo recordings of compound muscle action potential and electromyography amplitudes. However, BDNF hydrogel did not decrease lesion size or degeneration of cervical motor neuron soma, suggesting that its therapeutic mechanism of action was not neuroprotection within spinal cord. Interestingly, BDNF hydrogel significantly preserved diaphragm innervation by phrenic motor neurons (PhMNs), as assessed by detailed neuromuscular junction morphological analysis and retrograde PhMN labeling from diaphragm using cholera toxin B. Furthermore, BDNF hydrogel enhanced the serotonergic axon innervation of PhMNs that plays an important role in modulating PhMN excitability. Our findings demonstrate that local BDNF hydrogel delivery is a robustly effective and safe strategy to restore diaphragm function after SCI. In addition, we demonstrate novel therapeutic mechanisms by which BDNF can repair respiratory neural circuitry.SIGNIFICANCE STATEMENT Respiratory compromise is a leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). We used an innovative biomaterial-based drug delivery system in the form of a hydrogel that can be safely injected into the intrathecal space for achieving local delivery of brain-derived neurotrophic factor (BDNF) with controlled dosing and duration, while avoiding side effects associated with other delivery methods. In a clinically relevant rat model of cervical contusion-type SCI, BDNF hydrogel robustly and persistently improved diaphragmatic respiratory function by enhancing phrenic motor neuron (PhMN) innervation of the diaphragm neuromuscular junction and by increasing serotonergic innervation of PhMNs in ventral horn of the cervical spinal cord. These exciting findings demonstrate that local BDNF hydrogel delivery is a safe and robustly effective strategy to maintain respiratory function after cervical SCI.

Phrenic Motor Neuron Survival Below a Cervical Spinal Cord Injury

Cervical spinal injury (cSCI) severs descending brainstem projections to spinal respiratory motor neurons, paralyzing respiratory muscles caudal to injury. Spinal hemisection at C2 (C2Hx) has frequently been used as an experimental model of cSCI to study both spontaneous and induced functional recovery of respiratory and non‐respiratory somatic motor function. One assumption implicit in these studies is that C2Hx dennervates motor neurons below the injury, but does not cause significant motor neuron pathology or cell death. However, one recent study reported substantial, bilateral motor neuron death caudal to C2Hx (Satkunendrarajah et al, 2016); in this study, motor neurons were identified by ChAT immunoreactivity in the spinal ventral horn. We have also observed that retrograde labeling with Cholera toxin B fragment (CtB) is diminished in phrenic motor neurons following C2Hx (unpublished data) when the tracer was administered post‐C2Hx. However, these observations are potentially misleading since: 1) C2Hx may diminish ChAT immunoreactivity without motor neuron cell death; and 2) retrograde CtB transport may be diminished following injury due to motor neuron inactivity below the injury, leading to the false impression that phrenic motor neurons had died. Because phrenic motor neuron cell death following cSCI would have profound implications for studies of spontaneous and/or induced phrenic motor plasticity after C2Hx, we tested the hypothesis that C2Hx has minimal impact on phrenic motor neuron cell counts when phrenic motor neurons are retrogradely labeled with CtB prior to cSCI. Phrenic motor neurons were retrogradely labeled in intact adult Sprague Dawley rats via intrapleural injections of CtB. Rats were randomly assigned to one of four groups: 1) 2 week post‐C2Hx, 2) 8 week post‐C2Hx, or 3 & 4) age matched uninjured controls. Two weeks after CtB injection, rats in the C2Hx groups underwent left lateral C2Hx injury under isoflurane anesthesia. Rats were perfused at 2 or 8 wks post‐injury, and longitudinal sections (40 μm) from C3 to C8 were processed to reveal CtB and ChAT. CtB labeled phrenic motor neurons were quantified in serial sections; no loss of phrenic motor neurons was observed at either time post‐C2Hx versus spinally intact rats (p>0.05). Analysis of ChAT immunoreactivity is ongoing; however preliminary data suggest minimal changes in phrenic motor neuron ChAT expression following injury. Although we found little evidence for phrenic motor neuron cell death caudal to C2Hx, we cannot rule out greater cell loss in other motor neuron pools. Consequently, ChAT immunoreactivity is currently underway in non‐phrenic cervical motoneurons caudal to C2Hx. These findings have important implications for studies concerning plasticity in cervical motor systems following cervical spinal injury.Support or Funding InformationSupported by: K12HD055929 (EGR), RO1HL69064 (GSM), T32HD043730 (Allen), and grants from the DoD (GSM), the American Physiological Society (AH, JVS), the McNair Scholars Foundation (JVS), and the McKnight Brain Institute (GSM, MCC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of BDNF on Mitochondrial Morphology and Protein Expression in NSC‐34 Cells

Age related loss of larger phrenic motor neurons (PhMNs) may underlie diaphragm muscle sarcopenia. We found increased mitochondrial fragmentation in larger PhMNs, similar to that reported to be associated with motor neuron loss in amyotrophic lateral sclerosis (ALS). In rodents, we observed diminished brain‐derived neurotrophic factor (BDNF) signaling via its high affinity TrkB.FL receptor in older animals. TrkB.FL is expressed on the outer mitochondrial membrane, and in ALS models, BDNF/TrkB.FL signaling promotes motor neuron survival and mitochondrial fusion. In the present study, we hypothesized that enhancing BDNF/TrkB.FL signaling increases Mfn2 expression, thereby promoting mitochondrial fusion. Studies were conducted using a mouse motor neuron‐like cell line (NSC‐34), which were differentiated by serum depletion until neurite structures were observed. The differentiated NSC‐34 cells were treated with BDNF (12.5 ng/ml for 1 h) and then loaded with MitoTracker Green to image mitochondria using a Nikon A1R confocal microscope (60× oil immersion 1.4 NA). BDNF treatment increased both form factor and aspect ratio of mitochondria by ~ 20% and 15%, respectively, while also causing a threefold increase in expression of Mfn2, indicating mitochondrial fusion. These experiments were performed in six different trials with two replicates each. The observed relationship between BDNF/TrkB.FL signaling and mitochondrial fusion/fragmentation proteins supports our hypothesis. These results are consistent with diminished BDNF/TrkB.FL signaling underlying mitochondrial fragmentation and dysfunction in age‐related PhMN loss.Support or Funding InformationSupported by NIH grant AG44615This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diaphragm Muscle Function Following Mid‐Cervical Contusion Injury in Rats

Mid‐cervical spinal cord contusion injury results in tissue damage, disruption of spinal pathways, and motoneuron loss. Phrenic motoneurons located in C3–5 segments of the cervical spinal cord innervate the diaphragm muscle (DIAm), and unilateral C4 contusion results in loss of 40–50% of motoneurons ipsilateral to the injury (~25% of the total motoneuron pool). Over time after unilateral C4 spinal cord contusion injury, DIAm electromyography (EMG) increases both contralateral and ipsilateral to the side of injury, suggesting compensation due to an increased activation of the surviving motoneurons. However, whether C4 contusion impairs the ability of the DIAm to accomplish higher force motor behaviors is unknown. Transdiaphragmatic pressure (Pdi) was measured across motor behaviors over time after unilateral C4 contusion injury. Maximum Pdi (Pdimax) was elicited by bilateral phrenic nerve stimulation at 7 days post‐injury. We hypothesized that Pdimax is reduced following C4 mid‐cervical contusion injury, which will constrain the Pdi generated during different motor behaviors. Since ventilatory behaviors of the DIAm require <50% Pdimax, we further hypothesized that Pdi generated during ventilatory behaviors of the DIAm is not impaired after unilateral C4 mid‐cervical spinal cord contusion injury. In support of our hypothesis, we observed that Pdimax was reduced by ~25% after C4 mid‐cervical spinal cord contusion injury compared to a laminectomy control group. This decrease in Pdimax is consistent with the extent of phrenic motoneuron loss following contusion injury. We also found that during both eupnea (quiet breathing) and breathing stimulated by 10% O2 (hypoxia) and 5% CO2 (hypercapnia), Pdi generation was unimpaired by C4 mid‐cervical spinal cord contusion injury, again consistent with the lower force requirement of these ventilatory motor behaviors. Prior to injury, Pdi generated during airway occlusion was ~40% of Pdimax. One day following contusion injury, the Pdi amplitude during airway occlusion was reduced from ~30 cm H2O to ~20 cm, but this reduction was completely reversed by 7 days post‐injury. The reduction in Pdi amplitude at one day post‐injury cannot be attributed to the ~25% loss of phrenic motoneurons, and thus, may reflect a disruption of input to phrenic motoneurons or acute inflammation of their surrounding milieu. Over time after injury, changes in the balance between inhibition and excitation may result in recovery of higher‐force behaviors.Support or Funding InformationFunded by NIH R01‐HL096750 and T32‐HL105355This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spinal Conversion of Testosterone to Estradiol for the Expression of Phrenic Long‐term Facilitation in Adult Male Rats

Estrogen modulates the expression of neuroplasticity, including phrenic long‐term facilitation (pLTF), a persistent increase in phrenic nerve activity observed after acute exposure to intermittent hypoxia (AIH; three 5 min episodes of 10.5% O2, delivered at 5‐minute intervals). Though robustly expressed in adult males, adult female rats express pLTF only in the presence of adequate circulating estradiol (E2); removal of the ovaries, the primary source of circulating E2, abolishes pLTF. Acute, spinal application of membrane‐impermeable E2 (E2‐BSA) restores plasticity in ovariectomized female rats through a fast‐acting cellular mechanism involving membrane‐associated estrogen receptors, suggesting spinal E2 signaling may be important for pLTF expression in females. Previous studies indicate that E2 may play an important role in the expression of AIH‐induced pLTF in adult male rats as well. Specifically, gonadectomy blunts pLTF expression by reducing circulating testosterone, and systemic testosterone replacement restores pLTF. However, this testosterone‐induced restoration of pLTF is only effective in the presence of aromatase, an enzyme that converts testosterone to E2. Here we tested the hypothesis that the relevant conversion of testosterone to E2 necessary for expression of pLTF in male rats occurs in the spinal cord near phrenic motor neurons. We recorded phrenic nerve activity and pLTF expression in anesthetized, paralyzed and ventilated male Sprague‐Dawley rats (3–4 mos) in the presence or absence of intrathecally delivered Letrozole, an aromatase inhibitor, over the cervical spinal cord. We combined these data with immunohistochemical characterization of aromatase expression in the ventral cervical spinal cord. Collectively, these data provide important information regarding the necessity for spinal E2 signaling in the expression of AIH‐induced pLTF in male rats, and underscore the utility of this model to dissect the role of E2 signaling in expression of spinal neuroplasticity.Support or Funding InformationFunding: This work was supported by the University of Minnesota Division of Physical Therapy with a generous contribution from the University of Minnesota Department of Rehabilitation Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic afferent projections to the cervical spinal cord in spinal intact and spinal injured rats

The phrenic nerve contains both myelinated and unmyelinated afferent projections. Relatively little is known, however, regarding the spinal distribution of these sensory projections. Accordingly, our first goal was to create a map of phrenic afferent projections throughout the cervical spinal cord. Afferents were traced via prolonged phrenic nerve soak with Cascade Blue (CB) in adult Sprague‐Dawley rats. Spinal tissues were harvested, sectioned (40μm) from C2–C7, and processed with routine immunochemical methods. Extensive labeling of afferent projections was observed throughout the ipsilateral dorsal horn (laminae II and III) and intermediate grey region (laminae IV and V) in segments C2–C6. Phrenic afferent projections were crossed the spinal midline in 13% of the tissue sections, and were occasionally observed to reach the contralateral dorsal horn (laminae III). Robust phrenic motoneuron (PhrMN) labeling was observed from C3–C5, and PhrMN dendrites projected across the spinal midline in 30% of tissue sections from C3–C5. Our second objective was to determine if high cervical spinal cord injury (SCI) induced anatomical plasticity in the phrenic sensorimotor system. The phrenic nerve was traced using the same CB method in Sprague‐Dawley rats at 12 weeks following lateral hemisection of the C2 spinal cord. Preliminary analysis indicates that following SCI, phrenic afferents and PhrMN dendrites project across the spinal midline at approximately the same frequency as in spinal‐intact rats (p>0.36). However, we noted that the likelihood of observing phrenic afferent projections extending to the region of the ipsilateral phrenic motor nucleus (laminae VIII‐IX) was increased after SCI (p<0.05). By combining CB labeling with Choline acetyltransferase (ChAT) immunostaining, we verified the presence of laminae VIII‐IX phrenic afferents by observing CB‐positive and ChAT negative fibers. We conclude that phrenic sensory afferent projections 1) are prominent in the ipsilateral C3–C5 dorsal horn and intermediate grey region, 2) can project to the contralateral C3–C5 dorsal horn, and 3) undergo remodeling after high cervical SCI.Support or Funding InformationSPARC OT2 OD023854 (DDF), 1 R01 NS080180‐01A1 (DDF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Activation of Respiratory Muscles during Lower Thoracic High Frequency Spinal Cord Stimulation (HF‐SCS)

ObjectiveLower thoracic HF‐SCS (500 Hz, 1 mA) applied via a single stimulating electrode located on the dorsal surface of the spinal cord at the T9 level results in large positive airway pressures consistent with marked expiratory muscle activation. The purpose of the present study was to evaluate the effects of lower thoracic HF‐SCS on the ventral surface at this spinal level.MethodsIn 4 anesthetized dogs, HF‐SCS was applied via a stimulating electrode (4 mm disc) positioned on the ventral surface of the spinal cord at the T9 level. Airway pressure generation was monitored before and following spinal sections at the C8 (to eliminate potential diaphragm activation) and subsequently at the T6 (to eliminate potential intercostal muscle activation) levels.ResultsHF‐SCS (500 Hz, 1 mA) on the ventral surface, results in inspiratory airway pressure generation of 40 ± 3 cmH2O. Following complete C8 spinal section, HF‐SCS results in a significant reduction in negative airway pressure generation to 14 ± 1 cmH2O (p<0.05). Subsequent section at the T6 level, HF‐SCS results in a positive airway pressure of 10±1 cmH2O (p<0.05 when compared to before either section).ConclusionIn contrast to dorsal stimulation, lower thoracic ventral HF‐SCS (500 Hz, 1 mA), at T9 results in activation of pathways with connections to both the phrenic and inspiratory intercostal motoneuron pools resulting in the generation of the large negative airway pressures and minimal expiratory muscle activation. Under the experimental conditions of this study, neuronal pathways with connections to the inspiratory and expiratory motoneuron pools are each limited anatomically to specific regions of the spinal cord. These anatomic differences allow for separate activation of the inspiratory and expiratory muscles to restore function.Support or Funding InformationSupport: Merit Review Award I01‐RX‐001488 from the U.S. Department of Veterans Affairs, Rehabilitation Research and Development Service.Disclosure: Dr. DiMarco holds two U.S. patents for technology related to the content of this abstract: Method and Apparatus for Electrical Activation of the Expiratory Muscles to Restore Cough (5,999,855) and Bipolar Spinal Cord Stimulation to Activate the Expiratory Muscles to Restore Cough (8,751,004).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

5‐HT2B Receptor Expression in the Phrenic Motor Nucleus of Rats with Intrapleural CTB‐Saporin‐Induced Phrenic Motor Neuron Death

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death and decreased phrenic motor output. In this model, respiratory plasticity (acute intermittent hypoxia induced phrenic long‐term facilitation; pLTF) is enhanced following 7 days (d) of CTB‐SAP compared to control treatment. In contrast, pLTF in 28d CTB‐SAP treated rats is comparable to pLTF in controls, suggesting that the underlying mechanisms of plasticity at both time points may differ. pLTF in naïve rats predominately requires 5‐HT2 receptors; however, there is an alternative mechanism for pLTF that requires A2A receptors. Our preliminary data suggest that A2A receptors are increased in the phrenic motor nucleus of 7d, not 28d, CTB‐SAP treated rats (Craig et al., 2017), suggesting that 7d CTB‐SAP rats utilize the alternative mechanism for enhanced pLTF. However, it remains unknown how CTB‐SAP induced phrenic motor neuron death affects 5‐HT2A/B receptor expression, and if 5‐HT2A/B receptors are utilized for pLTF in CTB‐SAP treated rats. Here, we tested the hypothesis that 5‐HT2B receptor expression in the phrenic motor nucleus is decreased in 7d CTB‐SAP treated rats, but is not different in 28d CTB‐SAP treated rats when compared to controls using immunohistochemistry. 5‐HT2B receptor expression was studied in Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control). Our preliminary data indicate that following CTB‐SAP injection, total 5‐HT2B receptor positive pixels and C4 fractional area are decreased in the phrenic motor nucleus and non‐phrenic ventral horn at 7d, not 28d, vs. controls (n=3 for each group; p<0.05). This suggests 5‐HT2B receptors recover to control levels and are potentially utilized for pLTF in 28d CTB‐SAP treated rats. Future studies will be focused on whether 5‐HT2A receptor expression is similarly affected, whether 5‐HT2A/B receptor expression is impacted on other cell types (i.e., glial cells), and which 5‐HT receptor isoform is required for pLTF following CTB‐SAP‐induced phrenic motor neuron death. This project increases our understanding of the expression of receptors associated with respiratory plasticity and its implications for breathing following motor neuron death.Support or Funding InformationSupported by NIH K99/R00 HL119606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inflammation Differentially Impacts Phrenic Long‐term Facilitation (pLTF) in Rats with Motor Neuron Death Induced by Intrapleural CTB‐saporin Injections

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death, decreased phrenic motor output, and increased microglial cell number. Our previous results demonstrated unexpectedly that respiratory plasticity is not only observed 7 days (d) after CTB‐SAP delivery, but that it is actually enhanced compared to control treatments. At 28d, however, respiratory plasticity is comparable to control levels, suggesting that the underlying mechanisms of plasticity at both time points may differ. Because inflammation is a hallmark of all neurodegenerative diseases, and it is known to prevent the development of acute intermittent hypoxia‐induced phrenic long‐term facilitation (pLTF), we predicted that inflammation would impair pLTF in CTB‐SAP‐treated rats and thus, anti‐inflammatory drugs would enhance pLTF at both time points. Using anesthetized, paralyzed and ventilated CTB‐SAP‐treated adult Sprague Dawley rats, we studied acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) induced pLTF following intrathecal C4 delivery of the nonsteroidal anti‐inflammatory drug ketoprofen to rats that received bilateral, intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control) in rats treated 7d or 28d prior. Surprisingly, preliminary results indicate that ketoprofen attenuates pLTF in 7d CTB‐SAP‐treated rats, but enhances it in the 28d CTB‐SAP‐treated rats. These observations suggest that inflammation potentially contributes to pLTF in 7d CTB‐SAP‐treated rats, whereas it may undermine pLTF in 28d CTB‐SAP‐treated rats. Studies are underway to assess cervical spinal levels of both pro‐inflammatory and anti‐inflammatory and neurotrophic molecules at both time points, and their cellular source. Future studies will investigate the potential contributions of microglia to the mechanisms whereby inflammation differentially impacts pLTF in CTB‐SAP‐treated rats. Results from this project will increase our understanding of how inflammation and microglial activities influence respiratory plasticity following motor neuron death, and will have broad implications for therapies restoring ventilatory function in many disorders in which motor neuron death is associated.Support or Funding InformationNIH K99/R00 HL119606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

Although adenosine 2A (A2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A2A receptor siRNA injections. A2A receptor siRNA injections selectively knocked down A2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced phrenic motor facilitation. Upregulation of A2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A2A receptors for pMF are unknown. This is an important question since the physiological outcome of A2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89±2%). Intrapleural siRNA injections were used to selectively knock down A2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A2A receptor knock-down was verified by a ∼45% decrease in A2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P<0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A2A receptor agonist CGS21680 was greatly attenuated in siA2A (21%) versus siNT treated rats (147%; P<0.01). There were no significant effects of siA2A on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced pMF.

Systemic inflammation inhibits serotonin receptor 2-induced phrenic motor facilitation upstream from BDNF/TrkB signaling.

Although systemic inflammation induced by even a low dose of lipopolysaccharide (LPS, 100 μg/kg) impairs respiratory motor plasticity, little is known concerning cellular mechanisms giving rise to this inhibition. Phrenic motor facilitation (pMF) is a form of respiratory motor plasticity elicited by pharmacological agents applied to the cervical spinal cord, or by acute intermittent hypoxia (AIH; 3, 5-min hypoxic episodes); when elicited by AIH, pMF is known as phrenic long-term facilitation (pLTF). AIH consisting of moderate hypoxic episodes (mAIH, arterial Po2 = 35-55 mmHg) elicits pLTF via the Q pathway to pMF, a mechanism that requires spinal serotonin (5HT2) receptor activation and new brain-derived neurotrophic factor (BDNF) protein synthesis. Although mild systemic inflammation attenuates mAIH-induced pLTF via spinal p38 MAP kinase activation, little is known concerning how p38 MAP kinase activity inhibits the Q pathway. Here, we confirmed that 24 h after a low LPS dose (100 μg/kg ip), mAIH-induced pLTF is greatly attenuated. Similarly, pMF elicited by intrathecal cervical injections of 5HT2A (DOI; 100 μM; 3 × 6 μl) or 5HT2B receptor agonists (BW723C86; 100 μM; 3 × 6 μl) is blocked 24 h post-LPS. When pMF was elicited by intrathecal BDNF (100 ng, 12 μl), pMF was actually enhanced 24 h post-LPS. Thus 5HT2A/2B receptor-induced pMF is impaired downstream from 5HT2 receptor activation, but upstream from BDNF/TrkB signaling. Mechanisms whereby LPS augments BDNF-induced pMF are not yet known. NEW & NOTEWORTHY These experiments give novel insights concerning mechanisms whereby systemic inflammation undermines serotonin-dependent, spinal respiratory motor plasticity, yet enhances brain-derived neurotrophic factor (BDNF)/TrkB signaling in phrenic motor neurons. These insights may guide development of new strategies to elicit functional recovery of breathing capacity in patients with respiratory impairment by reducing (or bypassing) the impact of systemic inflammation characteristic of clinical disorders that compromise breathing.

Breathing: Motor Control of Diaphragm Muscle.

Breathing occurs without thought but is controlled by a complex neural network with a final output of phrenic motor neurons activating diaphragm muscle fibers (i.e., motor units). This review considers diaphragm motor unit organization and how they are controlled during breathing as well as during expulsive behaviors.

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Cell-type specific expression of constitutively-active Rheb promotes regeneration of bulbospinal respiratory axons following cervical SCI

Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality in individuals suffering from traumatic cervical spinal cord injury (SCI). Repair of CNS axons after SCI remains a therapeutic challenge, despite current efforts. SCI disrupts inspiratory signals originating in the rostral ventral respiratory group (rVRG) of the medulla from their phrenic motor neuron (PhMN) targets, resulting in loss of diaphragm function. Using a rat model of cervical hemisection SCI, we aimed to restore rVRG-PhMN-diaphragm circuitry by stimulating regeneration of injured rVRG axons via targeted induction of Rheb (ras homolog enriched in brain), a signaling molecule that regulates neuronal-intrinsic axon growth potential. Following C2 hemisection, we performed intra-rVRG injection of an adeno-associated virus serotype-2 (AAV2) vector that drives expression of a constitutively-active form of Rheb (cRheb). rVRG neuron-specific cRheb expression robustly increased mTOR pathway activity within the transduced rVRG neuron population ipsilateral to the hemisection, as assessed by levels of phosphorylated ribosomal S6 kinase. By co-injecting our novel AAV2-mCherry/WGA anterograde/trans-synaptic axonal tracer into rVRG, we found that cRheb expression promoted regeneration of injured rVRG axons into the lesion site, while we observed no rVRG axon regrowth with AAV2-GFP control. AAV2-cRheb also significantly reduced rVRG axonal dieback within the intact spinal cord rostral to the lesion. However, cRheb expression did not promote any recovery of ipsilateral hemi-diaphragm function, as assessed by inspiratory electromyography (EMG) burst amplitudes. This lack of functional recovery was likely because regrowing rVRG fibers did not extend back into the caudal spinal cord to synaptically reinnervate PhMNs that we retrogradely-labeled with cholera toxin B from the ipsilateral hemi-diaphragm. Our findings demonstrate that enhancing neuronal-intrinsic axon growth capacity can promote regeneration of injured bulbospinal respiratory axons after SCI, but this strategy may need to be combined with other manipulations to achieve reconnection of damaged neural circuitry and ultimately recovery of diaphragm function.

Phrenic motor neuron loss in aged rats.

Sarcopenia is the age-related reduction of muscle mass and specific force. In previous studies, we found that sarcopenia of the diaphragm muscle (DIAm) is evident by 24 mo of age in both rats and mice and is associated with selective atrophy of type IIx and IIb muscle fibers and a decrease in maximum specific force. These fiber type-specific effects of sarcopenia resemble those induced by DIAm denervation, leading us to hypothesize that sarcopenia is due to an age-related loss of phrenic motor neurons (PhMNs). To address this hypothesis, we determined the number of PhMNs in young (6 mo old) and old (24 mo old) Fischer 344 rats. Moreover, we determined age-related changes in the size of PhMNs, since larger PhMNs innervate type IIx and IIb DIAm fibers. The PhMN pool was retrogradely labeled and imaged with confocal microscopy to assess the number of PhMNs and the morphometry of PhMN soma and proximal dendrites. In older animals, there were 22% fewer PhMNs, a 19% decrease in somal surface area, and a 21% decrease in dendritic surface area compared with young Fischer 344 rats. The age-associated loss of PhMNs involved predominantly larger PhMNs. These results are consistent with an age-related denervation of larger, more fatigable DIAm motor units, which are required primarily for high-force airway clearance behaviors. NEW & NOTEWORTHY Diaphragm muscle sarcopenia in rodent models is well described in the literature; however, the relationship between sarcopenia and frank phrenic motor neuron (MN) loss is unexplored in these models. We quantify a 22% loss of phrenic MNs in old (24 mo) compared with young (6 mo) Fischer 344 rats. We also report reductions in phrenic MN somal and proximal dendritic morphology that relate to decreased MN heterogeneity in old compared with young Fischer 344 rats.

Intraspinal microstimulation for respiratory muscle activation

A complex propriospinal network is synaptically coupled to phrenic and intercostal motoneurons, and this makes it difficult to predict how gray matter intraspinal microstimulation (ISMS) will recruit respiratory motor units. We therefore mapped the cervical and high thoracic gray matter at locations which ISMS activates diaphragm (DIA) and external intercostal (EIC) motor units. Respiratory muscle electromyography (EMG) was recorded in anesthetized female spinally intact adult rats while a stimulating electrode was advanced ventrally into the spinal cord in 600μm increments. At each depth, single biphasic stimuli were delivered at 10–90μA during both the inspiratory and expiratory phase independently. Twenty electrode tracks were made from C2-T1 at medial and lateral gray matter locations. During inspiration, ISMS evoked DIA and EIC activity throughout C2-T1 gray matter locations, with mutual activation occurring at 17±9% of sites. During inspiratory phase ISMS the average latency for DIA activation was 4.40±0.70ms. During the expiratory phase, ISMS-induced DIA activation required electrodes to be in close proximity to the phrenic motoneuron pool, and average activation latency was 3.30±0.50ms. We conclude that appropriately targeted ISMS can co-activate DIA and EIC motor units, and endogenous respiratory drive has a powerful impact on ISMS-induced respiratory motor unit activation. The long latency diaphragm motor unit activation suggests the presence of a complex propriospinal network that can modulate phrenic motor output.

Cyclooxygenase enzyme activity does not impair respiratory motor plasticity after one night of intermittent hypoxia

Although inflammation is prevalent in many clinical disorders challenging breathing, we are only beginning to understand the impact of inflammation on neural mechanisms of respiratory control. We recently demonstrated one form of respiratory motor plasticity is extremely sensitive to even mild inflammation induced by a single night (8 h) of intermittent hypoxia (IH-1), mimicking aspects of obstructive sleep apnea. Specifically, phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (AIH) is abolished by IH-1, but restored by high doses of the non-steroidal anti-inflammatory drug, ketoprofen. Since a major target of ketoprofen is cyclooxygenase (COX) enzymes, we tested the involvement of COX in IH-1 suppression of pLTF using the selective COX inhibitor NS-398. Systemic COX inhibition (3 mg/kg, i.p., 3 h before AIH) had no effect on pLTF in normoxia treated rats (76 ± 40% change from baseline, n = 6), and did not restore pLTF in IH-1 treated rats (–9 ± 7% baseline, n = 6). Similarly, spinal COX inhibition (27 mM, 12 μl, i.t.) had no effect on pLTF in normoxic rats (76 ± 34% baseline, n = 7), and did not significantly restore pLTF after IH-1 (37 ± 18% baseline, n = 7). COX-2 protein is expressed in identified phrenic motor neurons of both normoxia and IH-1 exposed rats, but immunolabeling was minimal in surrounding microglia; IH-1 had no discernable effect on COX-2 immunoreactivity. We conclude that the inflammatory impairment of pLTF by IH-1 is independent of COX enzyme activity or upregulated COX-2 expression.

Topoisomerase IIβ Selectively Regulates Motor Neuron Identity and Peripheral Connectivity through Hox/Pbx-Dependent Transcriptional Programs.

Vital motor functions, such as respiration and locomotion, rely on the ability of spinal motor neurons (MNs) to acquire stereotypical positions in the ventral spinal cord and to project with high precision to their peripheral targets. These key properties of MNs emerge during development through transcriptional programs that dictate their subtype identity and connectivity; however, the molecular mechanisms that establish the transcriptional landscape necessary for MN specification are not fully understood. Here, we show that the enzyme topoisomerase IIβ (Top2β) controls MN migration and connectivity. Surprisingly, Top2β is not required for MN generation or survival but has a selective role in columnar specification. In the absence of Top2β, phrenic MN identity is eroded, while other motor columns are partially preserved but fail to cluster to their proper position. In Top2β-/- mice, peripheral connectivity is impaired as MNs exhibit a profound deficit in terminal branching. These defects likely result from the insufficient activation of Hox/Pbx-dependent transcriptional programs as Hox and Pbx genes are downregulated in the absence of Top2β. Top2β mutants recapitulate many aspects of Pbx mutant mice, such as MN disorganization and defects in medial motor column (MMC) specification. Our findings indicate that Top2β, a gene implicated in neurodevelopmental diseases such as autism spectrum disorders, plays a critical, cell-specific role in the assembly of motor circuits.