ABSTRACT
A major complication with spinal cord injury (SCI) is the development of spasticity, a clinical symptom of hyperexcitability within the spinal H-reflex pathway. We have previously demonstrated a common structural motif of dendritic spine dysgenesis associated with hyperexcitability disorders after injury or disease insults to the CNS. Here, we used an adeno-associated viral (AAV)-mediated Cre-Lox system to knockout Rac1 protein expression in motor neurons after SCI. Three weeks after AAV9-Cre delivery into the soleus/gastrocnemius of Rac1-"floxed" adult mice to retrogradely infect spinal alpha-motor neurons, we observed significant restoration of RDD and reduced H-reflex excitability in SCI animals. Additionally, viral-mediated Rac1 knockdown reduced presence of dendritic spine dysgenesis on motor neurons. In control SCI animals without Rac1 knockout, we continued to observe abnormal dendritic spine morphology associated with hyperexcitability disorder, including an increase in mature, mushroom dendritic spines, and an increase in overall spine length and spine head size. Taken together, our results demonstrate that viral-mediated disruption of Rac1 expression in ventral horn motor neurons can mitigate dendritic spine morphological correlates of neuronal hyperexcitability, and reverse hyperreflexia associated with spasticity after SCI. Finally, our findings provide evidence of a putative mechanistic relationship between motor neuron dendritic spine dysgenesis and SCI-induced spasticity.
Subject(s)
Anterior Horn Cells/metabolism , Depression/metabolism , Gene Knockout Techniques/methods , H-Reflex/genetics , Neuropeptides/metabolism , Spinal Cord Injuries/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Dendritic Spines/metabolism , Dependovirus/genetics , Depression/genetics , Disease Models, Animal , Female , Locomotion/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Spasticity/metabolism , Neuronal Plasticity/genetics , Neuropeptides/genetics , Spinal Cord Injuries/genetics , rac1 GTP-Binding Protein/geneticsABSTRACT
BACKGROUND: The Neurodata Without Borders data standard (NWB) unifies diverse modalities of neurophysiology data in a single format. Integrating NWB with a database unleashes its full potential to promote collaboration, standardize analyses, capitalize on historical data, and ensures data integrity by maintaining process transparency. NWB database technology is the bedrock of analytical systems used by academic leaders including the Allen Institute and the International Brain Laboratory. Here we present the benefits of incorporating NWB design principles in a big data analytics application. NEW METHOD: Data standards and databases are the foundation of big data analytics. To demonstrate the benefits of using these systems together, we implemented NWB in Jupyter notebooks using DataJoint to streamline database operations. RESULTS: We demonstrate the utility of combining the NWB with DataJoint in a Jupyter-based electronic lab journal. We convert open-field behavioral data (using X, Y coordinates) to NWB format and process it with a DataJoint pipeline. Additional notebooks demonstrate working NWB files, data sharing, combining data from diverse sources, and retrospective analyses with data query filtering techniques. COMPARISON WITH EXISTING METHODS: NWB describes how to structure and store neurophysiology data and is streamlined for research settings. In contrast to other data standards, combining NWB with DataJoint's database interface can dramatically increase data analytical capabilities. CONCLUSIONS: The joint use of NWB with DataJoint transforms traditional laboratory datasets and workflows. Our Jupyter notebooks showcase the analytical and collaborative advantages of adopting big data analytics and can be tailored to other modalities by researchers interested in evaluating NWB.
Subject(s)
Information Dissemination , Software , Data Science , Retrospective Studies , WorkflowABSTRACT
Neuropathic pain is an intractable medical condition with few or no options for effective treatment. Emerging evidence shows a strong structure-function relationship between dendritic spine dysgenesis and the presence of neuropathic pain. Postmortem tissue analyses can only imply dynamic structural changes associated with injury-induced pain. Here, we profiled the in vivo dynamics of dendritic spines over time on the same superficial dorsal horn (lamina II) neurons before and after peripheral nerve injury-induced pain. We used a two-photon, whole-animal imaging paradigm that permitted repeat imaging of the same dendritic branches of these neurons in C57/Bl6 Thy1-YFP male mice. Our study demonstrates, for the first time, the ongoing, steady-state changes in dendritic spine dynamics in the dorsal horn associated with peripheral nerve injury and pain. Ultimately, the relationship between altered dendritic spine dynamics and neuropathic pain may serve as a structure-based opportunity to investigate mechanisms of pain following injury and disease.SIGNIFICANCE STATEMENT This work is important because it demonstrates for the first time: (1) the powerful utility of intravital study of dendritic spine dynamics in the superficial dorsal horn; (2) that nerve injury-induced pain triggers changes in dendritic spine steady-state behavior in the spinal cord dorsal horn; and (3) this work opens the door to further investigations in vivo of spinal cord dendritic spine dynamics in the context of injury and disease.
Subject(s)
Dendritic Spines/pathology , Peripheral Nerve Injuries/pathology , Animals , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence, Multiphoton , Peripheral Nerve Injuries/physiopathology , Spinal Cord Dorsal Horn/pathology , Spinal Cord Dorsal Horn/physiopathologyABSTRACT
Burn injuries and associated complications present a major public health challenge. Many burn patients develop clinically intractable complications, including pain and other sensory disorders. Recent evidence has shown that dendritic spine neuropathology in spinal cord sensory and motor neurons accompanies central nervous system (CNS) or peripheral nervous system (PNS) trauma and disease. However, no research has investigated similar dendritic spine neuropathologies following a cutaneous thermal burn injury. In this retrospective investigation, we analyzed dendritic spine morphology and localization in alpha-motor neurons innervating a burn-injured area of the body (hind paw). To identify a molecular regulator of these dendritic spine changes, we further profiled motor neuron dendritic spines in adult mice treated with romidepsin, a clinically approved Pak1-inhibitor, or vehicle control at two postburn time points: Day 6 immediately after treatment, or Day 10 following drug withdrawal. In control treated mice, we observed an overall increase in dendritic spine density, including structurally mature spines with mushroom-shaped morphology. Pak1-inhibitor treatment reduced injury-induced changes to similar levels observed in animals without burn injury. The effectiveness of the Pak1-inhibitor was durable, since normalized dendritic spine profiles remained as long as 4 days despite drug withdrawal. This study is the first report of evidence demonstrating that a second-degree burn injury significantly affects motor neuron structure within the spinal cord. Furthermore, our results support the opportunity to study dendritic spine dysgenesis as a novel avenue to clarify the complexities of neurological disease following traumatic injury.
Subject(s)
Burns/physiopathology , Chronic Pain/physiopathology , Motor Neurons/physiology , Neuronal Plasticity , Spinal Cord/physiopathology , Animals , Burns/complications , Burns/drug therapy , Chronic Pain/drug therapy , Chronic Pain/etiology , Dendritic Spines/drug effects , Dendritic Spines/metabolism , Dendritic Spines/physiology , Depsipeptides/pharmacology , Depsipeptides/therapeutic use , Female , Hot Temperature , Male , Mice , Mice, Inbred C57BL , Motor Neurons/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Reflex , Spinal Cord/drug effects , p21-Activated Kinases/antagonists & inhibitorsABSTRACT
Neuropathic pain is a significant complication following spinal cord injury (SCI) with few effective treatments. Drug development for neuropathic pain often fails because preclinical studies do not always translate well to clinical conditions. Identification of biological characteristics predictive of disease state or drug responsiveness could facilitate more effective clinical translation. Emerging evidence indicates a strong correlation between dendritic spine dysgenesis and neuropathic pain. Because dendritic spines are located on dorsal horn neurons within the spinal cord nociceptive system, dendritic spine remodeling provides a unique opportunity to understand sensory dysfunction after SCI. In this study, we provide support for the postulate that dendritic spine profiles can serve as biomarkers for neuropathic pain. We show that dendritic spine profiles after SCI change to a dysgenic state that is characteristic of neuropathic pain in a Rac1-dependent manner. Suppression of the dysgenic state through inhibition of Rac1 activity is accompanied by attenuation of neuropathic pain. Both dendritic spine dysgenesis and neuropathic pain return when inhibition of Rac1 activity is lifted. These findings suggest the utility of dendritic spines as structural biomarkers for neuropathic pain.