Research PaperAccessory respiratory muscles enhance ventilation in ALS model mice and are activated by excitatory V2a neurons
Introduction
Amyotrophic lateral sclerosis (ALS) patients and animal models have the remarkable capacity to maintain relatively normal ventilation, despite progressive denervation and dysfunction of the diaphragm (Barneoud et al., 1997, Cappello et al., 2012, Hegedus et al., 2007, Kennel et al., 1996, Valdez et al., 2012), until late in disease progression when ventilation sharply declines (Hardiman, 2011, Nichols et al., 2013, Tankersley et al., 2007). Respiratory failure is the leading cause of death in ALS patients (Hardiman, 2011) and understanding how the respiratory system compensates - and ultimately fails to compensate - for the decline in motor function will be critical in the development of therapeutic interventions to prolong ventilator independence and improve the duration and quality of life of ALS patients.
Multiple compensatory mechanisms likely cooperate to maintain ventilation in ALS (Johnson and Mitchell, 2013, Nichols et al., 2013). One likely example, and the focus of this study, is the compensatory recruitment of inspiratory accessory respiratory muscles (ARMs). The diaphragm is the main inspiratory muscle in healthy mammals at rest. However, inspiratory ARMs, including the trapezius, scalenes, sternocleidomastoid, and external intercostal muscles are typically active during high ventilatory demand, such as exercise, to expand and stabilize the chest wall and enhance respiratory function (Sieck and Gransee, 2012). ARMs can also be recruited for breathing when diaphragm function is impaired, such as in ALS, spinal cord injury, and muscular dystrophy (Johnson and Mitchell, 2013, Pinto and de Carvalho, 2008, Smittkamp et al., 2010), and are sufficient to fully drive respiration in patients with diaphragmatic paralysis (Bennett et al., 2004). Remarkably, one study showed that ALS patients that recruit ARMs at rest sleep better and survive longer than patients with similar diaphragm dysfunction that do not recruit ARMs (Arnulf et al., 2000). It is not clear when during disease progression resting ARM activity begins or how long it might last. We use a unique combination of electromyography (via implanted telemetry devices) and whole body plethysmography to measure ARM activity and ventilation in non-anesthetized, unrestrained mice in the SOD1G93A model of ALS for which the onset and timing of disease progression is predictable, consistent, and well established.
Despite the importance of ARMs for maintaining breathing in a variety of physiological and pathological states, little is known about the neural circuits that drive ARM activity or how they might be altered by ALS pathology. An important step in understanding the circuits that control ARMs is to identify neuron types capable of driving or regulating inspiratory ARM activity. The V2a class of glutamatergic neuron in the spinal cord and brainstem has direct and indirect influences on respiratory and non-respiratory motor neurons (Azim et al., 2014, Bouvier et al., 2015, Bretzner and Brownstone, 2013, Crone et al., 2008, Crone et al., 2012, Crone et al., 2009, Zhong et al., 2010). V2a neurons comprise the majority of glutamatergic reticulospinal neurons in the medial reticular formation (mRF) (Bouvier et al., 2015, Bretzner and Brownstone, 2013). A subset of these neurons may be driven in a speed dependent manner based on the observation that they can be activated by the mesencephalic locomotor region (Bretzner and Brownstone, 2013). In addition, a population of V2a in the mRF have direct projections to the ventral respiratory column and provide excitatory drive to central respiratory circuits essential for normal frequency and regularity of breathing in neonatal mice (Crone et al., 2012). The potential role of V2a neurons in adult respiratory circuits has not yet been tested. Furthermore, spinal V2a neurons, located in the intermediate laminae, are required for the speed-dependent recruitment of locomotor pattern generator neurons as well as for maintaining consistent frequency and amplitude of motor burst activity during locomotion (Ampatzis et al., 2014, Crone et al., 2008, Crone et al., 2009, Kimura et al., 2013, Ljunggren et al., 2014). Because inspiratory ARM activity normally parallels locomotor activity and V2a neurons are important for speed-dependent activation of motor circuits, we hypothesize that a subset of V2a neurons could provide excitatory drive to coordinate and recruit ARM motor neurons (or premotor neurons) for breathing.
The goals of this study are to: 1) characterize the onset, extent and duration of ARM recruitment at rest during ALS-like disease progression in SOD1G93A mice, 2) examine the potential of V2a neurons to drive ARM activity in healthy mice, 3) determine if V2a neurons are adversely affected in SOD1G93A mice. Understanding how neural circuits control ARM activity will provide the necessary foundation for developing therapies targeting ARM circuits to improve breathing in ALS, spinal cord injury, and other pathological conditions.
Section snippets
Animals
All procedures were performed according to National Institutes of Health guidelines and approved by Cincinnati Children's Hospital Medical Center Animal Care and Use Committee. Plethysmography and/or electromyography was performed on adult male SOD1G93A transgenic mice (B6.Cg-Tg (SOD1*G93 A)1Gur/J; Stock #004,435 Jackson Laboratory, Bar Harbor, ME), adult male non-transgene carrier controls, and adult V2a-CHRM3 mice. V2a-CHRM3 mice were generated by first breeding ROSAPNP-tTA/+ (Stock #008,600
Increased accessory respiratory muscle recruitment in SOD1G93A mice
We evaluated activity of accessory respiratory muscles (ARMs) during ALS-like disease progression by implanting telemetry devices (e.g. radio transmitters) subcutaneously into male SOD1G93A mice or age and gender matched wildtype controls. Electromyography (EMG) leads were inserted into trapezius and scalene muscles. These devices record EMG activity in freely behaving animals from many motor units in the same muscle simultaneously and do not distinguish firing activity of individual motor
The frequency of ARM recruitment changes throughout disease progression
We demonstrate for the first time that accessory respiratory muscles are recruited for breathing at rest until late stages of disease progression in a mouse model of ALS. ARM activity is first apparent at the onset of limb weakness and increases in frequency over a period in which critical ventilation parameters are maintained (Tankersley et al., 2007), despite gradual diaphragm denervation (Barneoud et al., 1997, Kennel et al., 1996, Nichols et al., 2015). At late disease stages there is a
Conclusions
Our findings demonstrate that accessory respiratory muscles are recruited for breathing at rest during early stages of disease progression in the SOD1G93A mouse model of ALS. We also show that ARM recruitment at rest fails to occur at late stages of disease, even though the same ARMs are used for other behaviors, such as grooming and head and neck movements. Further, we identify a glutamatergic neuron class (V2a) in the spinal cord and brainstem whose activity is sufficient to drive ARM
Conflict of Interest Statement
The authors declare no conflicts of interest.
Acknowledgements
The authors thank Kelly Silnes and Lauren Kirgis for technical assistance. The authors also thank Roger Cornwall and Athanasia Nikolaou for assistance with our surgical approach and Steve Danzer and Raymund Pun for assistance with telemetry device implantation. This work was supported by an ALS Association Starter Grant (T7EMDY), an Emerging Investigator Award (2013) from FightSMA! and the Gwendolyn Strong Foundation, a Cincinnati Children's Hospital Research Foundation Trustee Award (2015),
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