Fogarty, Matthew J.

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

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

During development, GABA and glycine play major trophic and synaptic roles in the establishment of the neuromotor system. In this review, we summarise the formation, function and maturation of GABAergic and glycinergic synapses within neuromotor circuits during development. We take special care to discuss the differences in limb and respiratory neuromotor control. We then investigate the influences that GABAergic and glycinergic neurotransmission has on two major developmental neuromotor disorders: Rett syndrome and spastic cerebral palsy. We present these two syndromes in order to contrast the approaches to disease mechanism and therapy. While both conditions have motor dysfunctions at their core, one condition Rett syndrome, despite having myriad symptoms, has scientists focused on the breathing abnormalities and their alleviation—to great clinical advances. By contrast, cerebral palsy remains a scientific quagmire or poor definitions, no widely adopted model and a lack of therapeutic focus. We conclude that the sheer abundance of diversity of inhibitory neurotransmitter targets should provide hope for intractable conditions, particularly those that exhibit broad spectra of dysfunction—such as spastic cerebral palsy and Rett syndrome.

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.

AbstractSpinal cord hemisection at C2 (C2SH), sparing the dorsal column is widely used to investigate the effects of reduced phrenic motor neuron (PhMN) activation on diaphragm muscle (DIAm) function, with reduced DIAm activity on the injured side during eupnoea. Following C2SH, recovery of DIAm EMG activity may occur spontaneously over subsequent days/weeks. Various strategies have been effective at improving the incidence and magnitude of DIAm recovery during eupnoea, but little is known about the effects of C2SH on transdiaphragmatic pressure (Pdi) during other ventilatory and non‐ventilatory behaviours. We employ SPG302, a novel type of pegylated benzothiazole derivative, to assess whether enhancing synaptogenesis (i.e., enhancing spared local connections) will improve the incidence and the magnitude of recovery of DIAm EMG activity and Pdi function 14 days post‐C2SH. In anaesthetised Sprague‐Dawley rats, DIAm EMG and Pdi were assessed during eupnoea, hypoxia/hypercapnia and airway occlusion prior to surgery (C2SH or sham), immediately post‐surgery and at 14 days post‐surgery. In C2SH rats, 14 days of DMSO (vehicle) or SPG302 treatments (i.p. injection) occurred. At the terminal experiment, maximum Pdi was evoked by bilateral phrenic nerve stimulation. We show that significant EMG and Pdi deficits are apparent in C2SH compared with sham rats immediately after surgery. In C2SH rats treated with SPG302, recovery of eupneic, hypoxia/hypercapnia and occlusion DIAm EMG was enhanced compared with vehicle rats after 14 days. Treatment with SPG302 also ameliorated Pdi deficits following C2SH. In summary, SPG302 is an exciting new therapy to explore for use in spinal cord injuries. imageKey points Despite advances in our understanding of the effects of cervical hemisection (C2SH) on diaphragm muscle (DIAm) EMG activity, very little is understood about the impact of C2SH on the gamut of ventilatory and non‐ventilatory transdiaphragmatic pressures (Pdi). Recovery of DIAm activity following C2SH is improved using a variety of approaches, but very few pharmaceuticals have been shown to be effective. One way of improving DIAm recovery is to enhance the amount of latent local spared connections onto phrenic motor neurons. A novel pegylated benzothiazole derivative enhances synaptogenesis in a variety of neurodegenerative conditions. Here, using a novel therapeutic SPG302, we show that 14 days of treatment with SPG302 ameliorated DIAm EMG and Pdi deficits compared with vehicle controls. Our results show that SPG302 is a compound with very promising potential for use in improving functional outcomes post‐spinal cord injury.

Abstract Diaphragm muscle (DIAm) motor units comprise a phrenic motor neuron (PhMN), the phrenic nerve and the muscle fibers innervated, with the size of PhMNs and axons characteristic of motor unit type. Smaller PhMNs and their axons comprise slow (type S) and fatigue‐resistant (type FR) DIAm motor units, while larger PhMNs and their axons comprise more fatigable (type FF) motor units. With aging, we have shown a loss of larger PhMNs, consistent with selective atrophy of type IIx/IIb DIAm fibers and reduced maximum DIAm force. In the present study, we hypothesized that with aging there is a loss of larger myelinated phrenic α motor axons. Female and male young (6 months) and old (24 months) Fischer 344 rats were studied. PhMNs were retrogradely labeled by intrapleural injection of 488‐conjugated CTB. The phrenic nerves were excised ~1 cm from the DIAm insertion and mounted in resin, and phrenic α motor axons were delineated based on size (i.e., >4 μm diameters). In older rats, the number of larger PhMNs and larger phrenic α motor axons were reduced. There were no differences in non‐α axons. In addition, there was evidence of demyelination of larger phrenic α motor axons in older rats. Together, these findings are consistent with the selective age‐related vulnerability of larger PhMNs and denervation of type FF motor units, which may underlie DIAm sarcopenia.

Two weeks of diaphragm (DIAm) inactivity imposed by C2SH caused reduced mitochondrial volume density, mitochondrial fragmentation, and a concomitant reduction of SDH max in type I and IIa DIAm fibers on the lesioned side. Type I and IIa DIAm fibers were far more sensitive to inactivation than type IIx/IIb fibers, which exhibited little pathology. Our results indicate that mitochondria in DIAm fibers are plastic in response to varying levels of activity.

Developmental motor control dysfunctions, including spastic cerebral palsy (sCP) often have respiratory components. Spa mutant mice exhibit a spastic phenotype closely resembling sCP symptoms. Using the spa mouse model of spastic cerebral palsy (sCP), we quantified transdiaphragmatic pressure deficits, diaphragm muscle weakness, and fiber type-specific atrophy, improving our understanding of respiratory dysfunctions in sCP.

Type I and IIa diaphragm muscle (DIAm) fibers comprise fatigue resistant slow and fast motor units that are recruited to accomplish breathing. In contrast, type IIx/IIb fibers comprise more fatigable fast motor units that are infrequently recruited only during airway protective and straining behaviors. The activity and energetic demand for type I and IIa DIAm fibers is markedly increased compared to type IIx/IIb fibers. Cervical spinal cord hemisection at C 2 (C 2 SH) removes excitatory drive for inspiratory activity of the DIAm and thereby imposing prolonged inactivity of type I and IIa fibers. We hypothesize that mitochondrial structure and function in type I and IIa DIAm fibers adapt in response to imposed inactivity induced by spinal cord hemisection at C 2 (C 2 SH). After C 2 SH, inactivity was confirmed by the absence of DIAm EMG, and after 14 days, and the effect of inactivity on mitochondrial structure and function was assessed in type identified fibers. Mitochondria in DIAm fibers were labeled using MitoTracker Green and imaged in 3‐D by fluorescence confocal microscopy. Mitochondrial volume density and morphometry were analyzed using ImageJ. In alternate serial sections of the same DIAm fibers, the maximum velocity of the succinate dehydrogenase reaction (SDH max ) was determined using a quantitative histochemical technique. Overall, mitochondrial volume density was higher in type I and IIa DIAm fibers and mitochondria were more filamentous compared to type IIx/IIb fibers. Inactivity reduced mitochondrial volume density in type I and IIa DIAm fibers and induced mitochondrial fragmentation. The SDH max values normalized for fiber volume and for mitochondrial volume were both higher in type I and IIa DIAm fibers compared to type IIx/IIb fibers. After 14 days of inactivity induced by C 2 SH, SDH max (normalized for fiber volume and for mitochondrial volume) decreased in type I and IIa. These results are consistent with mitochondrial structural and functional adaptions to the marked reduction in the energetic demand of type I and IIa DIAm fibers imposed by C 2 SH.

The diaphragm (DIAm) and tibialis anterior (TA) muscles comprise both fatigue resistant (type I and IIa) and fatigable (type IIx/IIb) muscle fibers. Type I and IIa fibers in the DIAm are very frequently recruited for breathing, whereas in the TA, type I and IIa fibers are recruited for balance and locomotion. More fatigable type IIx/IIb fibers are only infrequently recruited for higher force behaviors in both the DIAm and TA. The repetitive activation of the DIAm during breathing is associated with a duty cycle of 40‐50%, making the DIAm one of, if not the most active muscle in the body ‐ much more active than the TA. These differences in activation history lead to differences in energetic demands between the DIAm and TA. We hypothesized that type I and IIa fibers in the DIAm exhibit a greater mitochondrial volume density and respiratory capacity (SDH max ) compared to type I and IIa fibers in the TA. Muscle segments from both DIAm and TA were dissected from adult female and male Sprague‐Dawley rats (~300 g), pinned to cork at optimal sarcomere length and rapidly frozen in melting isopentane. Subsequently, transverse serial sections of muscle fibers were cut at 10 µm thickness. In a series of transverse sections, muscle fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. In an alternate series of sections of the same muscle fibers, mitochondria were labeled using MitoTracker Green. In a third series of alternate sections, the maximum velocity of the succinate dehydrogenase reaction (SDH max ) was measured using a quantitative histochemical assay in which accumulation of the reaction product, nitroblue tetrazolium diformazan (NBT dfz ), was measured as a change in optical density (OD) in muscle fibers every 15 s for a 10‐min period. Muscle fiber sections were imaged in 3D using an Olympus Fluoview confocal microscope (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. In the mitoTracker labeled sections, each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI= SA 3 /16µ 2 V 2 ) using ImageJ and NIS elements. Mitochondrial volume density and morphometry were analyzed using ImageJ. The mitochondrial volume density and SDH max were greater in type I and IIa fibers of the DIAm compared to the TA. On the other hand, mitochondrial volume density and SDH max of type IIx/IIb fibers were comparable between the DIAm and TA. These results support mitochondrial structural and functional adaptations in type I and IIa DIAm fibers based on activation history and greater energetic demand.