Techniques and Approaches: From Molecules to Circuits to Whole Animal Physiology

The goal of the Crone Lab is to develop novel treatment strategies targeting neural circuits to restore breathing following injury or disease. The lab uses a variety of techniques to identify neurons important for the control of respiratory muscles and identify molecules in those neurons that have the potential to serve as drug targets to improve breathing.

We use mouse models of disease (ALS, muscular dystrophy, epilepsy) or injury (spinal cord injury) to investigate how breathing is altered and/or how the nervous system compensates for impaired respiratory function. A combination of transgenic mouse and viral tools such as chemogenetics (e.g., DREADDs), optogenetics, conditional knock-outs, neuron ablation, etc. are used to alter the activity or functions of specific classes of neurons in order to determine how they contribute to the control of respiratory muscles in healthy animals as well as following disease or injury.

We measure respiration using electromyography (EMG) to measure the activity of respiratory muscles and/or plethysmography to assess ventilation in awake or anesthetized mice. RNA sequencing is used to better understand the diversity of gene expression within spinal neurons, determine how they are affected by disease and injury at the molecular level, and to identify potential drug targets to improve breathing.

Current Projects

Our current research focuses on understanding how spinal neurons could be targeted to restore breathing after spinal cord injury, how accessory respiratory muscles are used to compensate for disease and injury, and how we might prevent sudden death in epilepsy (SUDEP).

Harnessing Spinal Network Plasticity to Restore Diaphragm Function Following Spinal Cord Injury

Cervical spinal cord injuries can disrupt communication between the brainstem centers that generate the breathing rhythm and respiratory motor neurons in the spinal cord, leading to paralysis of breathing muscles. Some (but not all) patients will spontaneously recover breathing function over time, due in part to changes in spinal circuits that control respiratory motor neurons.

We found that altering the firing activity of one class of spinal neuron (V2a neurons) can restore inspiratory activity to a previously paralyzed diaphragm in a mouse model of cervical spinal cord injury.

Current work focuses on identifying genes expressed by V2a neurons that are important for respiratory circuit plasticity and could serve as drug targets to improve breathing following injury.

Project Details

  • Funding: Craig H. Neilsen Foundation “Targeting propriospinal neurons to improve breathing following injury.”

Control of Accessory Respiratory Muscles

Although the diaphragm is the primary muscle used for breathing, additional muscles in the chest, neck and abdomen (accessory respiratory muscles) are recruited to enhance ventilation when we run, cough, sigh or take deep breaths. These muscles are also critical for maintaining ventilation and preventing respiratory infections when diaphragm function is impaired, as occurs in patients with neuromuscular disease or following spinal cord injury.

We found that the V2a class of neurons activates accessory respiratory muscles when needed as well as keeps them inactive at rest. 

We also discovered that these neurons degenerate in a mouse model of amyotrophic lateral sclerosis (ALS), suggesting that the loss of compensation by accessory respiratory muscles may contribute to ventilatory failure at late stages of disease.

Our lab is currently working to further elucidate circuits that control respiratory muscles in order to prevent ventilator dependence following disease or injury.

Project Details

  • Funding: NIH R01 “Spinal Circuitry for Ventilatory Control and Compensation.”

Exploring the Diversity of Propriospinal Neurons and Their Role in Recovery From Injury and Disease

We use transgenic and viral approaches to label and manipulate the activity of specific subsets of propriospinal neurons so that we can learn about their function(s) and their role in recovery from disease and injury. For example, we identified a subset of propriospinal neurons that degenerates early in disease progression in a mouse model of Amyotrophic Lateral Sclerosis. We also use single cell RNA sequencing and bioinformatic analyses to better understand what makes propriospinal neurons different from each other. We found that different subsets of propriospinal neurons show different changes in gene expression during recovery from spinal cord injury. Our lab is currently using this data to identify signaling molecules and pathways that could promote neural circuit plasticity and improve breathing following disease and injury.

Project Details

  • Funding
    • NIH R01 “Spinal Circuitry for Ventilatory Control and Compensation.”
    • Craig H. Neilsen Foundation “Targeting propriospinal neurons to improve breathing following injury.”

Preventing Respiratory Deficits Leading to Sudden Death in Epilepsy (SUDEP)

Each year, about 1 in 1,000 people with epilepsy die of SUDEP. The incidence climbs to more than 1 in 150 for epilepsy patients with uncontrolled seizures. The causes are unknown but are thought to be due to cardiorespiratory dysfunction.

In collaboration with the Gross Lab, we are investigating breathing abnormalities observed in a mouse model of SUDEP using implantable telemetry devices to chronically measure diaphragm activity (EMG) and detect seizures (EEG).

Our goal is to determine which signaling pathway(s) and brain regions are responsible for the breathing abnormalities and whether drugs targeting specific pathways can prevent breathing abnormalities and SUDEP.

Project Details

  • Funding
    • Citizens United for Research in Epilepsy (CURE) Foundation “PI3K signaling as a novel disease mechanism-based target to prevent or reduce SUDEP."
    • NIH R21 “Assessing the contribution of altered PI3K signaling to breathing abnormalities and sudden death in epilepsy.”