Benevides Ethan S, Thakre Prajwal P, Rana Sabhya, Sunshine Michael D, Jensen Victoria N, Oweiss Karim, Fuller David D
Department of Physical Therapy, University of Florida, Gainesville, United States.
Breathing Research and Therapeutics Center, University of Florida, Gainesville, United States.
Elife. 2025 Jun 2;13:RP97846. doi: 10.7554/eLife.97846.
Impaired respiratory motor output contributes to morbidity and mortality in many neurodegenerative diseases and neurologic injuries. We investigated if expressing designer receptors exclusively activated by designer drugs (DREADDs) in the mid-cervical spinal cord could effectively stimulate phrenic motor output to increase diaphragm activation. Two primary questions were addressed: (1) does effective DREADD-mediated diaphragm activation require focal expression in phrenic motoneurons (vs. non-specific mid-cervical expression), and (2) can this method produce a sustained increase in inspiratory tidal volume? Wild-type (C57Bl/6) 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 neuronal DREADD expression in the C4 ventral horns, targeting phrenic motoneurons. Diaphragm electromyograms (EMG) were recorded in isoflurane-anesthetized spontaneously breathing mice at 4-9 weeks post-AAV delivery. The 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 ChAT-Cre rats were conducted to determine if spinal DREADD activation could increase inspiratory tidal volume during spontaneous breathing, assessed using whole-body plethysmography 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 tidal volume. Subsequently, phrenic nerve recordings performed under urethane anesthesia confirmed that J60 evoked a >200% increase in inspiratory output. We conclude that targeting mid-cervical spinal DREADD expression to the phrenic motoneuron pool enables ligand-induced, sustained increases in phrenic motor output and tidal volume. Further development of this technology may enable application to clinical conditions associated with impaired diaphragm activation and hypoventilation.
呼吸运动输出受损在许多神经退行性疾病和神经损伤中会导致发病和死亡。我们研究了在颈髓中部表达仅由设计药物激活的设计受体（DREADDs）是否能有效刺激膈神经运动输出以增加膈肌激活。我们探讨了两个主要问题：（1）有效的DREADD介导的膈肌激活是否需要在膈运动神经元中局部表达（相对于非特异性的颈髓中部表达），以及（2）这种方法能否使吸气潮气量持续增加？野生型（C57Bl/6）和ChAT-Cre小鼠接受了双侧脊髓内（C4）注射编码hM3D(Gq)兴奋性DREADD的腺相关病毒（AAV）。在野生型小鼠中，这在整个颈髓中部腹角产生了非特异性的DREADD表达。在ChAT-Cre小鼠中，使用一种依赖Cre的病毒构建体来驱动C4腹角神经元的DREADD表达，以靶向膈运动神经元。在AAV注射后4 - 9周，在异氟烷麻醉下自主呼吸的小鼠中记录膈肌肌电图（EMG）。DREADD配体JHU37160（J60）使两组的吸气EMG爆发出现双侧、持续（>1小时）增加；ChAT-Cre小鼠中的相对增加更大。在ChAT-Cre大鼠中进行了额外实验，以确定脊髓DREADD激活是否能在自主呼吸期间增加吸气潮气量，这是在无麻醉情况下使用全身体积描记法评估的。在脊髓内（C4）注射驱动依赖Cre的hM3D(Gq)表达的AAV三到四个月后，静脉注射J60导致潮气量持续（>30分钟）增加。随后，在氨基甲酸乙酯麻醉下进行的膈神经记录证实，J60使吸气输出增加>200%。我们得出结论，将颈髓中部的DREADD表达靶向到膈运动神经元池可使配体诱导的膈神经运动输出和潮气量持续增加。这项技术的进一步发展可能使其应用于与膈肌激活受损和通气不足相关的临床情况。
