We hypothesized that considerable force reserve exists for the diaphragm muscle (DIAm) to generate transdiaphragmatic pressures (Pdi) necessary to sustain ventilation. In rats, we measured Pdi and DIAm EMG activity during different ventilatory (eupnea and hypoxia (10% O2) – hypercapnia (5% CO2)) and non-ventilatory (airway occlusion and sneezing induced by intranasal capsaicin) behaviors. Compared to maximum Pdi (Pdimax - generated by bilateral phrenic nerve stimulation), the Pdi generated during eupnea (21±2%) and hypoxia-hypercapnia (28±4%) were significantly less (P<0.0001) than that generated during airway occlusion (63±4%) and sneezing (94±5%). The Pdi generated during spontaneous sighs was 62±5% of Pdimax. Relative DIAm EMG activity (root mean square [RMS] amplitude) paralleled the changes in Pdi during different ventilatory and non-ventilatory behaviors (r2=0.78; p<0.0001). These results support our hypothesis of a considerable force reserve for the DIAm to accomplish ventilatory behaviors. A model for DIAm motor unit recruitment predicted that ventilatory behaviors would require activation of only fatigue resistant units.

Early in the development of COPD, diaphragm fiber contractile function is impaired. Our data suggest that enhanced diaphragm protein degradation through the ubiquitin-proteasome pathway plays a role in loss of contractile protein and, consequently, failure of the diaphragm to generate force.

. Effects of voluntary activity and genetic selection on aerobic capacity in house mice (Mus domesticus). J. Appl. Physiol. 84(1): 69-76, 1998.-An animal model was developed to study effects on components of exercise physiology of both ''nature'' (10 generations of genetic selection for high voluntary activity on running wheels) and ''nurture'' (7-8 wk of access or no access to running wheels, beginning at weaning). At the end of the experiment, mice from both wheel-access groups were significantly lighter in body mass than mice from sedentary groups. Within the wheel-access group, a statistically significant, negative relationship existed between activity and final body mass. In measurements of maximum oxygen consumption during forced treadmill exercise (V O 2 max ), mice with wheel access were significantly more cooperative than sedentary mice; however, trial quality was not a significant predictor of individual variation in V O 2 max . Nested two-way analysis of covariance demonstrated that both genetic selection history and access to wheels had significant positive effects on V O 2 max . A 12% difference in V O 2 max existed between wheelaccess selected mice, which had the highest mass-corrected V O 2 max , and sedentary control mice, which had the lowest. The respiratory exchange ratio at V O 2 max was also significantly lower in the wheel-access group. Our results suggest the existence of a possible genetic correlation between voluntary activity levels (behavior) and aerobic capacity (physiology).

We hypothesized that metabolic adaptations to muscle inactivity are most pronounced when neurotrophic influence is disrupted. In rat diaphragm muscle (Dia(m)), 2 wk of unilateral denervation or tetrodotoxin nerve blockade resulted in a reduction in succinate dehydrogenase (SDH) activity of type I, IIa, and IIx fibers (approximately 50, 70, and 24%, respectively) and a decrease in SDH variability among fibers (approximately 63%). In contrast, inactivity induced by spinal cord hemisection at C2 (ST) resulted in much less change in SDH activity of type I and IIa fibers (approximately 27 and 24%, respectively) and only an approximately 30% reduction in SDH variability among fibers. Actomyosin adenosinetriphosphatase (ATPase) activities of type I, IIx, and IIb fibers in denervated and tetrodotoxin-treated Dia(m) were reduced by approximately 20, 45, and 60%, respectively, and actomyosin ATPase variability among fibers was approximately 60% lower. In contrast, only actomyosin ATPase activity of type IIb fibers was reduced (approximately 20%) in ST Dia(m). These results suggest that disruption of neurotrophic influence has a greater impact on muscle fiber metabolic properties than inactivity per se.

In the adult rat, there is a general correspondence between the sizes of motoneurons, motor units, and muscle fibers that has particular functional importance in motor control. During early postnatal development, after the establishment of singular innervation, there is rapid growth of diaphragm muscle (Dia(m)) fibers. In the present study, the association between Dia(m) fiber growth and changes in phrenic motoneuron size (both somal and dendritic) was evaluated from postnatal day 21 (D21) to adulthood. Phrenic motoneurons were retrogradely labeled with fluorescent tetramethylrhodamine dextran (3,000 MW), and motoneuron somal volumes and surface areas were measured using three-dimensional confocal microscopy. In separate animals, phrenic motoneurons retrogradely labeled with choleratoxin B-fragment were visualized using immunocytochemistry, and dendritic arborization was analyzed by camera lucida. Between D21 and adulthood, Dia(m) fiber cross-sectional area increased by approximately 164% overall, with the growth of type II fibers being disproportionate to that of type I fibers. There was also substantial growth of phrenic motoneurons ( approximately 360% increase in total surface area), during this same period, that was primarily attributable to an expansion of dendritic surface area. Comparison of the distribution of phrenic motoneuron surface areas between D21 and adults suggests the establishment of a bimodal distribution that may have functional significance for motor unit recruitment in the adult rat.

Studies of motoneuron plasticity during development or in response to injury or disease rely on the ability to correctly identify motoneurons innervating specific muscle groups. Commonly, injections of retrograde tracer molecules into a target muscle or into a transected nerve are used to label specific motoneuron pools. However, intramuscular injection does not consistently label all motoneurons in the target pool; and either injection site may involve additional surgical procedures and muscle or nerve perturbations. For instance, retrograde labeling of phrenic motoneurons by injection into the diaphragm muscle is commonly employed in studies of plasticity in respiratory motor control. Diaphragm intramuscular injection involves a laparotomy, and this additional surgery may limit the ability to conduct labeling studies particularly in small animals. In the present study, we provide validation of a novel method for phrenic motoneuron labeling using intrapleural injection of Alexa 488-conjugated cholera toxin subunit B. Only phrenic motoneurons were labeled in the cervical spinal cord as verified by co-staining with rhodamine-conjugated dextran injected into the diaphragm muscle or applied via phrenic nerve dip. Thoracic intercostal motoneurons and some dorsal root ganglia neurons were also labeled by intrapleural injection, but there was no evidence of transsynaptic labeling. Phrenic motoneuron labeling was not present if the phrenic nerve was transected prior to intrapleural injection. This novel method is ideally suited for morphological studies and analyses of mRNA expression in isolated phrenic motoneurons using techniques such as laser capture microdissection.

A C 2 cervical spinal cord hemisection (SH) interrupts descending inspiratory-related drive to phrenic motoneurons located between C 3 and C 5 in rats, paralyzing the ipsilateral hemidiaphragm muscle. There is gradual recovery of rhythmic diaphragm muscle activity ipsilateral to cervical spinal cord injury over time, consistent with neuroplasticity and strengthening of spared, contralateral descending premotor input to phrenic motoneurons. Brainderived neurotrophic factor (BDNF) signaling through the tropomyosin related kinase receptor subtype B (TrkB) plays an important role in neuroplasticity following spinal cord injury. We hypothesized that 1) increasing BDNF/TrkB signaling at the level of the phrenic motoneuron pool by intrathecal BDNF delivery enhances functional recovery of rhythmic diaphragm activity after SH, and 2) inhibiting BDNF/ TrkB signaling by quenching endogenous neurotrophins with the soluble fusion protein TrkB-Fc or by knocking down TrkB receptor expression in phrenic motoneurons using intrapleurallydelivered siRNA impair functional recovery after SH. Diaphragm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of SH and 3 days post-SH. After SH surgery in adult rats, an intrathecal catheter was placed at C 4 to chronically infuse BDNF or TrkB-Fc using an implanted mini-osmotic pump. At 14 days post-SH, all intrathecal BDNF treated rats (n=9) displayed recovery of ipsilateral hemidiaphragm EMG activity, compared to 3 out of 8 untreated SH rats (p < 0.01). During eupnea, BDNF treated rats exhibited 76 ± 17% of pre-SH root mean squared EMG vs. only 5 ± 3% in untreated SH rats (p < 0.01). In contrast, quenching endogenous BDNF with intrathecal TrkB-Fc treatment completely prevented functional recovery up to 14 days post-SH (n=7). Immunoreactivity of the transcription factor cAMP response element-binding protein (CREB), a downstream effector of TrkB signaling, increased in phrenic motoneurons following BDNF treatment (n=6) compared to artificial cerebrospinal fluid treatment (n=6; p < 0.001). Intrapleural injections of non-sense or TrkB siRNA were administered after SH to specifically target phrenic motoneurons. At 14 days post-SH, none out of 9 TrkB siRNA treated rats displayed functional recovery compared to 5 out of 9 non-sense siRNA treated rats. These results indicate that BDNF/TrkB signaling in phrenic motoneuron pool plays a critical role in functional recovery after cervical spinal cord injury.

Neurotrophins modulate acute and sustained synaptic plasticity. In cultured Xenopus laevis neuromuscular junctions, neurotrophins improve neuromuscular transmission. Whether this influence exists at the mammalian neuromuscular junction is unknown. We hypothesized that neurotrophins improve neuromuscular transmission at neuromuscular junctions of adult rat diaphragm muscle fibers. A diaphragm muscle-phrenic nerve preparation was used to determine the effects of brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and K252a [tyrosine kinase (Trk) receptor inhibitor] on the extent of neuromuscular transmission failure induced by repetitive nerve stimulation. We found significant enhancement of neuromuscular transmission with BDNF or NT-4 treatment, whereas K252a treatment worsened neuromuscular transmission. In contrast, diaphragm muscle contractile and fatigue properties were unaffected by neurotrophin or K252a treatment. These results demonstrate that BDNF and NT-4 improve synaptic transmission in the adult rat diaphragm muscle, likely in a Trk-dependent fashion. Neurotrophins may constitute a novel therapeutic target to improve neuromuscular function in the diaphragm.