Chemogenetic stimulation of phrenic motor output and diaphragm activity.

Impaired diaphragm activation contributes to morbidity and mortality in many neurodegenerative diseases and neurologic injuries. We conducted experiments to determine if expression of an excitatory DREADD (designer receptors exclusively activation by designer drugs) in the mid-cervical spinal cord would enable respiratory-related activation of phrenic motoneurons to increase diaphragm activation. Wild type (C57/bl6) and ChAT-Cre mice received bilateral intraspinal (C4) injections of an adeno-associated virus (AAV) encoding the hM3D(Gq) excitatory DREADD. In wild type mice, this produced non-specific DREADD expression throughout the mid-cervical ventral horn. In ChAT-Cre mice, a Cre-dependent viral construct was used to drive DREADD expression in C4 ventral horn motoneurons, targeting the phrenic motoneuron pool. Diaphragm EMG was recorded during spontaneous breathing at 6-8 weeks post-AAV delivery. The selective DREADD ligand JHU37160 (J60) caused a bilateral, sustained (>1 hr) increase in inspiratory EMG bursting in both groups; the relative increase was greater in ChAT-Cre mice. Additional experiments in a ChAT-Cre rat model were conducted to determine if spinal DREADD activation could increase inspiratory tidal volume (VT) during spontaneous breathing without anesthesia. Three to four months after intraspinal (C4) injection of AAV driving Cre-dependent hM3D(Gq) expression, intravenous J60 resulted in a sustained (>30 min) increase in VT assessed using whole-body plethysmography. Subsequently, direct nerve recordings confirmed that J60 evoked a >50% increase in inspiratory phrenic output. The data show that mid-cervical spinal DREADD expression targeting the phrenic motoneuron pool enables ligand-induced, sustained increases in the neural drive to the diaphragm. Further development of this technology may enable application to clinical conditions associated with impaired diaphragm activation and hypoventilation.

Influence of chronic hypoxia on the hypoxic ventilatory response of juvenile and adult rats.

Chronic hypoxia (CH) from birth attenuates the acute hypoxic ventilatory response (HVR) in rats and other mammals, but CH is often reported to augment the HVR in adult mammals. To test the hypothesis that this transition - from blunting to augmenting the HVR - occurs in the third or fourth postnatal week in rats, juvenile and adult rats were exposed to normobaric CH (12% O2) for 7 days and the HVR was assessed by whole-body plethysmography. No transition was observed, however, and the acute HVR was reduced by 61 - 85% across all ages studied. The failure to observe an augmented HVR in adult rats could not be explained by the substrain of Sprague Dawley rats used, the duration of the CH exposure, the order in which test gases were presented, the level of hypoxia used for CH and to assess the HVR, or the effects of CH on the metabolic response to hypoxia and the hypercapnic ventilatory response. A literature survey revealed several distinct patterns of ventilatory acclimatization to hypoxia (VAH) in adult rats, with most studies (77%) revealing a decrease or no change in the acute HVR after CH. In conclusion, the effects of CH on respiratory control are qualitatively similar across age groups, at least within the populations of Sprague Dawley rats used in the present study, and there does not appear to be one "typical" pattern for VAH in adult rats.

Targeting drug or gene delivery to the phrenic motoneuron pool.

Phrenic motoneurons (PhrMNs) innervate diaphragm myofibers. Located in the ventral gray matter (lamina IX), PhrMNs form a column extending from approximately the third to sixth cervical spinal segment. Phrenic motor output and diaphragm activation are impaired in many neuromuscular diseases, and targeted delivery of drugs and/or genetic material to PhrMNs may have therapeutic application. Studies of phrenic motor control and/or neuroplasticity mechanisms also typically require targeting of PhrMNs with drugs, viral vectors, or tracers. The location of the phrenic motoneuron pool, however, poses a challenge. Selective PhrMN targeting is possible with molecules that move retrogradely upon uptake into phrenic axons subsequent to diaphragm or phrenic nerve delivery. However, nonspecific approaches that use intrathecal or intravenous delivery have considerably advanced the understanding of PhrMN control. New opportunities for targeted PhrMN gene expression may be possible with intersectional genetic methods. This article provides an overview of methods for targeting the phrenic motoneuron pool for studies of PhrMNs in health and disease.

Optogenetic activation of the tongue in spontaneously breathing mice.

Inadequate tongue muscle activation contributes to dysarthria, dysphagia, and obstructive sleep apnea. Thus, treatments which increase tongue muscle activity have potential clinical benefit. We hypothesized that lingual injection of an adeno-associated virus (AAV) encoding channelrhodopsin-2 (ChR2) would enable light-induced activation of tongue motor units during spontaneous breathing. An AAV serotype 9 vector (pACAGW-ChR2-Venus-AAV9, 8.29 × 1011 vg) was injected to the posterior tongue in adult C57BL/6J mice. After 12 weeks, mice were anesthetized and posterior tongue electromyographic (EMG) activity was recorded during spontaneous breathing; a light source was positioned near the injection site. Light-evoked EMG responses increased with the intensity and duration of pulses. Stimulus trains (250 ms) evoked EMG bursts that were comparable to endogenous (inspiratory) tongue muscle activation. Histology confirmed lingual myofiber transgene expression. We conclude that intralingual AAV9-ChR2 delivery enables light evoked lingual EMG activity. These proof-of-concept studies lay the groundwork for clinical application of this novel approach to lingual therapeutics.

Chemogenetic activation of hypoglossal motoneurons in a mouse model of Pompe disease.

Pompe disease is a lysosomal storage disease resulting from absence or deficiency of acid α-glucosidase (GAA). Tongue-related disorders including dysarthria, dysphagia, and obstructive sleep apnea are common in Pompe disease. Our purpose was to determine if designer receptors exclusively activated by designer drugs (DREADDs) could be used to stimulate tongue motor output in a mouse model of Pompe disease. An adeno-associated virus serotype 9 (AAV9) encoding an excitatory DREADD (AAV9-hSyn-hM3D(Gq)-mCherry, 2.44 × 1010 vg) was administered to the posterior tongue of 5-7-wk-old Gaa null (Gaa-/-) mice. Lingual EMG responses to intraperitoneal injection of saline or a DREADD ligand (JHU37160-dihydrochloride, J60) were assessed 12 wk later during spontaneous breathing. Saline injection produced no consistent changes in lingual EMG. Following the DREADD ligand, there were statistically significant (P < 0.05) increases in both tonic and phasic inspiratory EMG activity recorded from the posterior tongue. Brainstem histology confirmed mCherry expression in hypoglossal (XII) motoneurons in all mice, thus verifying retrograde movement of the AAV9 vector. Morphologically, Gaa-/- XII motoneurons showed histological characteristics that are typical of Pompe disease, including an enlarged soma and vacuolization. We conclude that lingual delivery of AAV9 can be used to drive functional expression of DREADD in XII motoneurons in a mouse model of Pompe disease.NEW & NOTEWORTHY In a mouse model of Pompe disease, lingual injection of adeno-associated virus (AAV) serotype 9 encoding DREADD was histologically verified to produce transgene expression in hypoglossal motoneurons. Subsequent intraperitoneal delivery of a DREADD ligand stimulated tonic and phase tongue motor output.In a mouse model of Pompe disease, lingual injection of adeno-associated virus (AAV) serotype 9 encoding DREADD was histologically verified to produce transgene expression in hypoglossal motoneurons. Subsequent intravenous delivery of a DREADD ligand stimulated tonic and phase tongue motor output.

Optogenetic activation of the diaphragm.

Impaired diaphragm activation is common in many neuromuscular diseases. We hypothesized that expressing photoreceptors in diaphragm myofibers would enable light stimulation to evoke functional diaphragm activity, similar to endogenous bursts. In a mouse model, adeno-associated virus (AAV) encoding channelrhodopsin-2 (AAV9-CAG-ChR2-mVenus, 6.12 × 1011 vg dose) was delivered to the diaphragm using a minimally invasive method of microinjection to the intrapleural space. At 8-18 weeks following AAV injection, mice were anesthetized and studied during spontaneous breathing. We first showed that diaphragm electromyographic (EMG) potentials could be evoked with brief presentations of light, using a 473 nm high intensity LED. Evoked potential amplitude increased with intensity or duration of the light pulse. We next showed that in a paralyzed diaphragm, trains of light pulses evoked diaphragm EMG activity which resembled endogenous bursting, and this was sufficient to generate respiratory airflow. Light-evoked diaphragm EMG bursts showed no diminution after up to one hour of stimulation. Histological evaluation confirmed transgene expression in diaphragm myofibers. We conclude that intrapleural delivery of AAV9 can drive expression of ChR2 in the diaphragm and subsequent photostimulation can evoke graded compound diaphragm EMG activity similar to endogenous inspiratory bursting.

Gene delivery to the hypoglossal motor system: preclinical studies and translational potential.

Dysfunction and/or reduced activity in the tongue muscles contributes to conditions such as dysphagia, dysarthria, and sleep disordered breathing. Current treatments are often inadequate, and the tongue is a readily accessible target for therapeutic gene delivery. In this regard, gene therapy specifically targeting the tongue motor system offers two general strategies for treating lingual disorders. First, correcting tongue myofiber and/or hypoglossal (XII) motoneuron pathology in genetic neuromuscular disorders may be readily achieved by intralingual delivery of viral vectors. The retrograde movement of viral vectors such as adeno-associated virus (AAV) enables targeted distribution to XII motoneurons via intralingual viral delivery. Second, conditions with impaired or reduced tongue muscle activation can potentially be treated using viral-driven chemo- or optogenetic approaches to activate or inhibit XII motoneurons and/or tongue myofibers. Further considerations that are highly relevant to lingual gene therapy include (1) the diversity of the motoneurons which control the tongue, (2) the patterns of XII nerve branching, and (3) the complexity of tongue muscle anatomy and biomechanics. Preclinical studies show considerable promise for lingual directed gene therapy in neuromuscular disease, but the potential of such approaches is largely untapped.