Citations

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 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.

Type S and FR motor units, comprising smaller phrenic motor neurons (PhMNs) are regularly activated to perform indefatigable ventilatory requirements. By contrast, type FF motor units, comprising larger PhMNs, are infrequently activated to perform expulsive straining and airway defense maneuvers. This difference in activation history is mirrored in the mitochondrial volume density (MVD), with smaller PhMNs having higher MVD than larger PhMNs. In proximal dendrites, this trend was reversed, with larger PhMNs having higher MVD than smaller PhMNs, likely due to the maintenance requirements for the larger dendritic arbor of FF PhMNs.

Abstract Cerebral malaria (CM) is a severe complication arising from Plasmodium falciparum infection which results in high mortality. Lack of mechanistic insight into this pathology has resulted in ineffective therapeutic strategies. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM) our lab recapitulated hallmarks of human CM using small animal imaging, including blood-brain barrier (BBB) permeability, severe edema and microhemorrhage visible via T1 and T2-weighted MRI. Previously it had been demonstrated that infiltration of CD8 T cells with cytotoxic capacity is required to induce BBB permeability and ECM. The observation that not only infiltration, but cytotoxic killing is required provoked us to analyze differential MHC class I-restricted priming among discrete antigen presenting cell (APC) types. Using new MHC class I conditional knockout mice generated by our team, we addressed CD8 T cell priming and capacity to disrupt the BBB in ECM. Our findings revealed that mice lacking the H-2Kb MHC class I molecule expression in CD11c+ cells, but not LysM+ cells, were protected from the development of lethal ECM. Subsequently, conditional ablation of the H-2Db on these same subsets yielded similar results. Therefore, we demonstrate that both Db and Kb class I molecule expression by CD11c+ cells enable CD8 T cell priming that results in BBB permeability during ECM. In contrast, CD8 T cells primed by H-2Kb or H-2Db expressing LysM+ APCs are not capable of inducing BBB disruption. These results imply discrete APC subsets prime functionally distinct CD8 T cell responses in the CNS and modulate the outcome of cerebral malaria. This work begins to bridge the mechanistic gap and will impact future therapeutic strategies.

Abstract Cerebral malaria (CM) is a deadly neurological complication of Plasmodium falciparum infection resulting in high mortality or severe cognitive deficit. Current therapeutics are highly ineffective, namely due to drug resistance and lack of mechanistic insight into pathophysiology. CM is characterized by VEGF upregulation, disruption of blood-brain barrier (BBB), vascular permeability, and severe edema detectable by MRI. Using small animal MRI and the established animal model of CM Plasmodium berghei ANKA, we recapitulate these features of human disease. Here we show that regions of brain permeability during CM as identified by T1-weighted gadolinium-enhanced and T2-weighted MRI coincide with VEGF mRNA upregulation. Regions of analysis include granular and glomerular cell layers of main olfactory bulb and superior medullary brainstem. We determined through RNA in situ hybridization that neurons are the major cell type responsible for the increased VEGF production during CM. Interestingly, we identified another cell type found to minimally upregulate VEGF in close proximity to these neurons. These VEGF expressing cells may play a role in support or induction of the neuronal response. Intriguingly, astrocytes show no VEGF upregulation during this pathology. Overall, these findings suggest a novel role for neuronal-VEGF in promoting CNS pathology, and a putative mechanism of BBB disruption and vascular permeability during CM.

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 .

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 (Pdi max ) was elicited by bilateral phrenic nerve stimulation at 7 days post‐injury. We hypothesized that Pdi max 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% Pdi max , 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 Pdi max was reduced by ~25% after C4 mid‐cervical spinal cord contusion injury compared to a laminectomy control group. This decrease in Pdi max 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% O 2 (hypoxia) and 5% CO 2 (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 Pdi max . One day following contusion injury, the Pdi amplitude during airway occlusion was reduced from ~30 cm H 2 O 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 Information Funded by NIH R01‐HL096750 and T32‐HL105355 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Key points Motor units, comprising a motor neuron and the muscle fibre it innervates, are activated in an orderly fashion to provide varying amounts of force. A unilateral C2 spinal hemisection (C2SH) disrupts predominant excitatory input from medulla, causing cessation of inspiratory‐related diaphragm muscle activity, whereas higher force, non‐ventilatory diaphragm activity persists. In this study, we show a disproportionately larger loss of excitatory glutamatergic innervation to small phrenic motor neurons (PhMNs) following C2SH, as compared with large PhMNs ipsilateral to injury. Our data suggest that there is a dichotomy in the distribution of inspiratory‐related descending excitatory glutamatergic input to small vs. large PhMNs that reflects their differential recruitment. AbstractExcitatory glutamatergic input mediating inspiratory drive to phrenic motor neurons (PhMNs) emanates primarily from the ipsilateral ventrolateral medulla. Unilateral C2 hemisection (C2SH) disrupts this excitatory input, resulting in cessation of inspiratory‐related diaphragm muscle (DIAm) activity. In contrast, after C2SH, higher force, non‐ventilatory DIAm activity persists. Inspiratory behaviours require recruitment of only smaller PhMNs, whereas with more forceful expulsive/straining behaviours, larger PhMNs are recruited. Accordingly, we hypothesize that C2SH primarily disrupts glutamatergic synaptic inputs to smaller PhMNs, whereas glutamatergic synaptic inputs to larger PhMNs are preserved. We examined changes in glutamatergic presynaptic input onto retrogradely labelled PhMNs using immunohistochemistry for VGLUT1 and VGLUT2. We found that 7 days after C2SH there was an ∼60% reduction in glutamatergic inputs to smaller PhMNs compared with an ∼35% reduction at larger PhMNs. These results are consistent with a more pronounced impact of C2SH on inspiratory behaviours of the DIAm, and the preservation of higher force behaviours after C2SH. These results indicate that the source of glutamatergic synaptic input to PhMNs varies depending on motor neuron size and reflects different functional control – perhaps separate central pattern generator and premotor circuits. For smaller PhMNs, the central pattern generator for inspiration is located in the pre‐Bötzinger complex and premotor neurons in the ventrolateral medulla, sending predominantly ipsilateral projections via the dorsolateral funiculus. C2SH disrupts this glutamatergic input. For larger PhMNs, a large proportion of excitatory inputs appear to exist below the C2 level or from contralateral regions of the brainstem and spinal cord.

Glutamatergic (Glu) synaptic inputs at phrenic motor neurons (PhMNs) predominantly comprise descending axons from respiratory centers in the ventrolateral medulla. Descending synaptic inputs provide the drive for inspiratory as well as various other behaviors that result in higher force generation. We recently examined pre‐synaptic Glu terminals (VGLUT1 and VGLUT2‐positive) around the somal membrane of PhMNs grouped according to size using a robust confocal imaging‐based technique and semi‐automated image processing, and found an ~10% higher density of Glu terminals at PhMNs in the lower tertile of somal surface area. In parallel studies, we determined the density of AMPA and NMDA mRNA expression in PhMNs grouped according to size, and found that PhMNs in the lower tertile of somal surface area display ~25% higher density of AMPA (Gria2) and NMDA (Grin1) mRNA expression compared to PhMNs in the upper tertile of somal surface area. Assuming an orderly recruitment of motor units based on their intrinsic electrophysiological properties, PhMNs in the lower tertile of somal surface area are likely recruited first in order to accomplish lower force behaviors necessary for inspiration in normoxic as well as in hypoxic (10% O 2 ) and hypercapnic (5% CO 2 ) conditions. Thus, differences in glutamatergic neurotransmission across PhMNs of increasing size support the orderly recruitment of motor units, promoting recruitment of fatigue resistant units for inspiratory behaviors. In adult male rats, unilateral C2 hemisection (C2SH) abolishes inspiratory‐related diaphragm muscle (DIAm) activity ipsilateral to injury, but DIAm activity during higher force behaviors persists, reflecting the contribution of spared inputs, likely contralateral. Accordingly, we hypothesized that C2SH primarily disrupts Glu synaptic inputs to smaller PhMNs, whereas Glu synaptic inputs to larger PhMNs are preserved. By 7 days following unilateral C2SH, there was a ~45% reduction in ipsilateral Glu synaptic input to PhMNs compared to a ~20% reduction contralaterally. Ipsilateral to C2SH, there was an ~60% reduction in presynaptic Glu terminals at PhMNs in lower tertile of somal surface area compared to ~30% reduction at PhMNs in the upper tertile of size. Contralateral to C2SH, the reduction in presynaptic Glu terminals was similar across PhMNs grouped according to somal surface area. These results are consistent with a more pronounced effect of C2SH on inspiratory behaviors of the DIAm, and the preservation of higher force behaviors after C2SH. These results also indicate that 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, onto PhMNs recruited for motor behaviors with varying levels of force generation. Support or Funding Information NIH Grants R01 HL96750 and HL146114

Brain‐derived neurotrophic factor (BDNF) signaling via its high affinity tropomyosin‐related kinase sub‐type B (TrkB) receptor is widely implicated in regulating glutamatergic synaptic transmission in the context of neuroplasticity. At phrenic motor neurons innervating the main inspiratory muscle in mammals (the diaphragm muscle), glutamate is the main excitatory neurotransmitter and acts primarily via ionotropic AMPA and NMDA receptors. In various regions in the CNS, BDNF/TrkB signaling influences rapid phosphorylation of AMPA and NMDA receptor subunits, supporting a main role in neuroplasticity. BDNF/TrkB signaling also plays an important role across various models of injury and respiratory neuroplasticity. In addition, BDNF/TrkB signaling increases AMPA and NMDA receptor expression in the CNS, but whether this effect is also present in motor neurons is presently unknown. BDNF/TrkB signaling modulates motor neuron excitability, which may reflect changes in glutamatergic receptor expression. In the present study, AMPA and NMDA mRNA expression in retrogradely‐labeled phrenic motor neurons was measured using RNAscope in situ hybridization (ACDBio). Adult male rats were randomly assigned to control (untreated) and 11‐day treatment groups (12 ml/day) with intrathecal infusion of the fusion‐protein TrkB‐Fc or artificial cerebrospinal fluid (aCSF) in the region of the phrenic motor neuron pool. Intrathecal catheter tip position was verified at the terminal experimental at ~C4 segment, and the infusion rate was confirmed to restrict delivery to the cervical spinal cord. Expression of both AMPA and NMDA receptor mRNA was evident as puncta in phrenic motor neurons (identified by cholera toxin sub‐unit B – CTB‐labeling) as well as in other large neurons in the ventral horn. Our analysis of mRNA puncta density was restricted to clearly‐identified, retrogradely (CTB)‐labeled phrenic motor neurons containing the mid‐nuclear region in frozen, thin (10μm) longitudinal sections of the spinal cord. At least 25 phrenic motor neurons were stereologically selected for analysis in each animal. No differences in AMPA or NMDA mRNA density were evident between the untreated control and aCSF treated groups, and these results were combined. Across phrenic motor neurons in the control and aCSF groups, there was ~30% variance in AMPA and NMDA mRNA density within each animal, reflecting heterogeneity across motor neurons of varying size, expected to be recruited for the various levels of force production by the diaphragm muscle. Following TrkB‐Fc infusion, there was evidence of decreased AMPA mRNA density in phrenic motor neurons following but no change in NMDA mRNA density. These results support a role for BDNF/TrkB signaling in modulating glutamatergic receptor expression at phrenic motor neurons, which may thus contribute to the regulation of motor neuron excitability and neuroplasticity following injury or disease. Support or Funding Information NIH grants R01 HL96750 and R01 HL146114