Zhan, Wen‐Zhi

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMN) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMN. Neurotrophins play an important role in regulating synaptic function. Thus, we hypothesized that intrathecal brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic phrenic activity after C2 spinal cord hemisection (C2HS), whereas quenching neurotrophins with TrkB‐IgG impairs functional recovery. An intrathecal catheter was placed at C4 to chronically infuse artificial CSF, BDNF or TrkB‐Fc using an osmotic pump. DIAm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of C2HS and 3D post‐C2HS. In the artificial CSF group, functional recovery was evident in 33% of C2HS rats at 7D and 56% of rats at 14D post‐injury. Intrathecal BDNF treatment enhanced functional recovery, with 88% of rats at 7D and 100% at 14D post‐C2HS displaying rhythmic phrenic activity. In contrast, intrathecal TrkB‐Fc treatment completely prevented functional recovery up to 14D post‐C2HS. Our results demonstrate that BDNF/TrkB signaling at the level of the PMN pool is both sufficient and necessary for functional recovery of rhythmic phrenic activity after C2HS. Supported by NIH grant AR51173.

Myasthenia gravis is a disease of the neuromuscular junction (NMJ) associated with autoimmune destruction of the motor end‐plate. Inhibitors of acetylcholine (ACh) esterase are commonly used to mitigate symptoms by increasing ACh availability at the NMJ. Brain‐derived neurotrophic factor (BDNF) improves neuromuscular transmission in the rat diaphragm muscle (DIAm) by maintaining ACh release with repeated activation. We hypothesized that BDNF will mitigate the deficit of neuromuscular transmission in a passive transfer model of myasthenia gravis. Female Lewis rats were injected with vehicle, McAb1 (that does not induce myasthenia), and a myasthenogenic McAb3 antibody. Neuromuscular transmission failure (NMTF) was determined in DIAm‐phrenic nerve preparations by comparing the isometric force evoked by phrenic nerve stimulation and that evoked by superimposed direct muscle stimulation. After 2 min of repetitive stimulation, neuromuscular transmission was significantly impaired in myasthenic animals (NMTF: 80%) compared to vehicle and McAb1 animals (NMTF: ~60%). Exogenous BDNF treatment significantly improved neuromuscular transmission in all groups and NMTF in McAb3‐treated rats was no longer different from control or McAb1‐treated animals. We conclude that BDNF improves neuromuscular transmission in adult myasthenic rats. Supported by a Career Development Award from Mayo Clinic.

Neuregulin (NRG) is important for expression of acetylcholine (ACh) receptors during development of the neuromuscular junction (NMJ). NRG treatment significantly increases ACh‐induced currents in hippocampal neurons, but it is not known if NRG exerts a similar effect at NMJs. Diaphragm muscle (DIAm)‐phrenic nerve preparations from 7‐ and 14‐day old rats were used to determine the effects of NRG (heregulin β177–244) on neuromuscular transmission and DIAm contractile properties. Neuromuscular transmission failure (NMTF) was assessed by the difference in force elicited by repetitive phrenic nerve stimulation (0.2 ms pulses in 40 Hz, 330 ms trains) and superimposed, intermittent direct DIAm stimulation (1 ms pulses in 40 Hz, 330 ms trains applied every 15 seconds). NMTF was higher in 7‐day old compared to 14‐day old rats (76% vs. 65%; p<0.05). Exogenous NRG treatment significantly improved neuromuscular transmission in 7‐day old rats (NMTF: 52%; p<0.05), but not in 14‐day old rats (65%). Isometric DIAm contractile properties were not affected by NRG treatment in either age group. These results indicate that NRG has important synaptic effects during early postnatal development of the DIAm. Whether NRG effects on neuromuscular transmission result from acute changes in AChR expression or alternative mechanisms remains to be explored. Supported by NIH grant AR51173 and a Career Development Award from Mayo Clinic.

Brain-derived neurotrophic factor (BDNF) plays an important role in promoting neuroplasticity following unilateral C2 spinal hemisection (C2SH). BDNF was administered intrathecally in rats displaying lack of ipsilateral inspiratory-related diaphragm (DIAm) EMG activity after C2SH. Acute BDNF treatment (30 min) restored eupneic DIAm EMG activity in all treated animals to 78% ± 9% of pre-C2SH level. In addition, acute BDNF treatment increased DIAm EMG amplitude during hypoxia-hypercapnia but had no effect on EMG amplitude during sighs.

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

In previous studies, we found that after cervical spinal cord hemisection at C2 (SH), neuromuscular transmission improved in the rat diaphragm muscle (DIAm). We also found that exogenous brain‐derived neurotrophic factor (BDNF) treatment enhanced neuromuscular transmission, whereas inhibiting activation of its receptor (TrkB) worsened neuromuscular transmission. We hypothesized that expression of BDNF and/or its receptor TrkB at phrenic motoneurons would increase following SH. In the present study, adult male rats underwent SH or sham surgery. Phrenic motoneurons were labeled by injection of cholera toxin B into the DIAm and retrogradely‐labeled phrenic motoneurons were sampled by laser capture microdissection (LCM). BDNF and TrkB receptor (truncated – T1 and full length – FL) mRNA were measured in LCM‐microdissected motoneurons using quantitative real‐time RT‐PCR and expressed as a ratio to ribosomal S16 RNA. We found that BDNF mRNA expression in phrenic motoneurons increased by 3 days after SH (~2‐fold). Similarly, both TrkB.T1 and TrkB.FL mRNA increased by 3 days after SH (~17‐ and ~11‐fold, respectively). It is possible that the relative expression of neurotrophins and their receptors (truncated or full‐length) may determine the time‐course of restoration of phrenic activity following SH. Supported by NIH grants AR51173 and HL37680 and the Mayo Foundation.

DIAm motor units are normally recruited in an orderly fashion to accomplish different ventilatory and non‐ventilatory behaviors. Following a cervical spinal cord hemisection at C2 (SH), there is restoration of phrenic activity over time. We hypothesized that an orderly recruitment of DIAm motor units is maintained with the restoration of rhythmic DIAm electromyographic (EMG) activity following SH. A pair of electrode wires was implanted into the midcostal regions of the DIAm of adult male rats for chronic EMG recording. Three days later, these animals underwent SH surgery. We examined changes in integrated DIAm EMG activity across different motor behaviors (i.e., eupnea, hypercapnia, tracheal occlusion and fictive sneezing) assuming that DIAm EMG activity during fictive sneezing represents the maximum recruitment of DIAm motor units. As expected in controls, DIAm EMG activity increased from eupnea (~27% of max) to hypercapnia (5% CO 2; ~45% of max) to sustained tracheal occlusion (~60% of max). By 14 days after SH with restored rhythmic DIAm activity, the amplitude of EMG activity on the ipsilateral side was reduced, and the changes in EMG activity induced by increased ventilatory and non‐ventilatory drive were blunted, although an orderly recruitment of motor units was still observed. These results support a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury. Supported by NIH grants AR51173 and HL37680 and the Mayo Foundation.