Cervical Epidural Electrical Stimulation Increases Respiratory Activity through Somatostatin-Expressing Neurons in the Dorsal Cervical Spinal Cord in Rats.

We tested the hypothesis that dorsal cervical epidural electrical stimulation (CEES) increases respiratory activity in male and female anesthetized rats. Respiratory frequency and minute ventilation were significantly increased when CEES was applied dorsally to the C2-C6 region of the cervical spinal cord. By injecting pseudorabies virus into the diaphragm and using c-Fos activity to identify neurons activated during CEES, we found neurons in the dorsal horn of the cervical spinal cord in which c-Fos and pseudorabies were co-localized, and these neurons expressed somatostatin (SST). Using dual viral infection to express the inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADD), hM4D(Gi), selectively in SST-positive cells, we inhibited SST-expressing neurons by administering Clozapine N-oxide (CNO). During CNO-mediated inhibition of SST-expressing cervical spinal neurons, the respiratory excitation elicited by CEES was diminished. Thus, dorsal cervical epidural stimulation activated SST-expressing neurons in the cervical spinal cord, likely interneurons, that communicated with the respiratory pattern generating network to effect changes in ventilation.SIGNIFICANCE STATEMENT A network of pontomedullary neurons within the brainstem generates respiratory behaviors that are susceptible to modulation by a variety of inputs; spinal sensory and motor circuits modulate and adapt this output to meet the demands placed on the respiratory system. We explored dorsal cervical epidural electrical stimulation (CEES) excitation of spinal circuits to increase ventilation in rats. We identified dorsal somatostatin (SST)-expressing neurons in the cervical spinal cord that were activated (c-Fos-positive) by CEES. CEES no longer stimulated ventilation during inhibition of SST-expressing spinal neuronal activity, thereby demonstrating that spinal SST neurons participate in the activation of respiratory circuits affected by CEES. This work establishes a mechanistic foundation to repurpose a clinically accessible neuromodulatory therapy to activate respiratory circuits and stimulate ventilation.

Noninvasive measurement of sensory action currents in the cervical cord by magnetospinography.

OBJECTIVE: To obtain magnetic recordings of electrical activities in the cervical cord and visualize sensory action currents of the dorsal column, intervertebral foramen, and dorsal horn. METHODS: Neuromagnetic fields were measured at the neck surface upon median nerve stimulation at the wrist using a magnetospinography system with high-sensitivity superconducting quantum interference device sensors. Somatosensory evoked potentials (SEPs) were also recorded. Evoked electrical currents were reconstructed by recursive null-steering beamformer and superimposed on cervical X-ray images. RESULTS: Estimated electrical currents perpendicular to the cervical cord ascended sequentially. Their peak latency at C5 and N11 peak latency of SEP were well-correlated in all 16 participants (r = 0.94, p < 0.0001). Trailing axonal currents in the intervertebral foramens were estimated in 10 participants. Estimated dorsal-ventral electrical currents were obtained within the spinal canal at C5. Current density peak latency significantly correlated with cervical N13-P13 peak latency of SEPs in 13 participants (r = 0.97, p < 0.0001). CONCLUSIONS: Magnetospinography shows excellent spatial and temporal resolution after median nerve stimulation and can identify the spinal root entry level, calculate the dorsal column conduction velocity, and analyze segmental dorsal horn activity. SIGNIFICANCE: This approach is useful for functional electrophysiological diagnosis of somatosensory pathways.

Visualization of electrical activity in the cervical spinal cord and nerve roots after ulnar nerve stimulation using magnetospinography.

OBJECTIVE: To establish a method for magnetospinography (MSG) measurement after ulnar nerve stimulation and to clarify its characteristics. METHODS: Using a 132-channel magnetoneurography system with a superconducting quantum interference device, cervical MSG measurements were obtained for 10 healthy volunteers after stimulation of the ulnar nerve at the elbow and the wrist, and neural current distribution was calculated and superimposed on the cervical X-ray images. RESULTS: Neuromagnetic signals were obtained in all participants after applying the stimulus artifact removal algorithm. The measured magnetic field intensity after elbow stimulation was about twice that after wrist stimulation. Calculated neural currents flowed into the intervertebral foramina at C6/7 to T1/2 and propagated cranially along the spinal canal. The conduction velocity from the peak latency of inward currents at C5-C7 was 73.4 ± 19.6 m/s. CONCLUSIONS: We successfully obtained MSG measurements after ulnar nerve stimulation. The neural currents flowed into the spinal canal from more caudal segments after ulnar nerve stimulation compared with median nerve stimulation, and these MSG measurements were effective in examining the spinal tracts at C5/6/7. SIGNIFICANCE: This is the first report on the use of MSG to visualize electrical activity in the cervical spinal cord and nerve root after ulnar nerve stimulation.

Circulatory control of phrenic motor plasticity.

Acute intermittent hypoxia (AIH) elicits distinct mechanisms of phrenic motor plasticity initiated by brainstem neural network activation versus local (spinal) tissue hypoxia. With moderate AIH (mAIH), hypoxemia activates the carotid body chemoreceptors and (subsequently) brainstem neural networks associated with the peripheral chemoreflex, including medullary raphe serotonergic neurons. Serotonin release and receptor activation in the phrenic motor nucleus then elicits phrenic long-term facilitation (pLTF). This mechanism is independent of tissue hypoxia, since electrical carotid sinus nerve stimulation elicits similar serotonin-dependent pLTF. In striking contrast, severe AIH (sAIH) evokes a spinal adenosine-dependent, serotonin-independent mechanism of pLTF. Spinal tissue hypoxia per se is the likely cause of sAIH-induced pLTF, since local tissue hypoxia elicits extracellular adenosine accumulation. Thus, any physiological condition exacerbating spinal tissue hypoxia is expected to shift the balance towards adenosinergic pLTF. However, since these mechanisms compete for dominance due to mutual cross-talk inhibition, the transition from serotonin to adenosine dominant pLTF is rather abrupt. Any factor that compromises spinal cord circulation will limit oxygen availability in spinal cord tissue, favoring a shift in the balance towards adenosinergic mechanisms. Such shifts may arise experimentally from treatments such as carotid denervation, or spontaneous hypotension or anemia. Many neurological disorders, such as spinal cord injury or stroke compromise local circulatory control, potentially modulating tissue oxygen, adenosine levels and, thus, phrenic motor plasticity. In this brief review, we discuss the concept that local (spinal) circulatory control and/or oxygen delivery regulates the relative contributions of distinct pathways to phrenic motor plasticity.

Optogenetic surface stimulation of the rat cervical spinal cord.

Electrical intraspinal microstimulation (ISMS) at various sites along the cervical spinal cord permits forelimb muscle activation, elicits complex limb movements and may enhance functional recovery after spinal cord injury. Here, we explore optogenetic spinal stimulation (OSS) as a less invasive and cell type-specific alternative to ISMS. To map forelimb muscle activation by OSS in rats, adeno-associated viruses (AAV) carrying the blue-light sensitive ion channels channelrhodopsin-2 (ChR2) and Chronos were injected into the cervical spinal cord at different depths and volumes. Following an AAV incubation period of several weeks, OSS-induced forelimb muscle activation and movements were assessed at 16 sites along the dorsal surface of the cervical spinal cord. Three distinct movement types were observed. We find that AAV injection volume and depth can be titrated to achieve OSS-based activation of several movements. Optical stimulation of the spinal cord is thus a promising method for dissecting the function of spinal circuitry and targeting therapies following injury. NEW & NOTEWORTHY Optogenetics in the spinal cord can be used both for therapeutic treatments and to uncover basic mechanisms of spinal cord physiology. For the first time, we describe the methodology and outcomes of optogenetic surface stimulation of the rat spinal cord. Specifically, we describe the evoked responses of forelimbs and address the effects of different adeno-associated virus injection paradigms. Additionally, we are the first to report on the limitations of light penetration through the rat spinal cord.

Biomechanical Effects on Cervical Spinal Cord and Nerve Root Following Laminoplasty for Ossification of the Posterior Longitudinal Ligament in the Cervical Spine: A Comparison Between Open-Door and Double-Door Laminoplasty Using Finite Element Analysis.

Many clinical case series have reported the predisposing factors for C5 palsy and have presented comparisons of the two types of laminoplasty. However, there have been no biomechanical studies focusing on cervical spinal cord and nerve root following laminoplasty. The purpose of this study is to investigate biomechanical changes in the spinal cord and nerve roots following the two most common types of laminoplasty, open-door and double-door laminoplasty, for cervical ossification of the posterior longitudinal ligament (OPLL). A finite element (FE) model of the cervical spine and spinal cord with nerve root complex structures was developed. Stress changes in the spinal cord and nerve roots, posterior shift of the spinal cord, and displacement of the cervical nerve roots were analyzed with two types of cervical laminoplasty models for variations in the degree of canal occupying ratio and shape of the OPLL. The shape and degree of spinal cord compression caused by the OPLL had more influence on the changes in stress, posterior shift of the spinal cord, and displacement of the nerve root than the type of laminoplasty. The lateral-type OPLL resulted in imbalanced stress on the nerve roots and the highest nerve root displacement. Type of laminoplasty and shape and degree of spinal cord compression caused by OPLL were found to influence the changes in stress and posterior displacement of the cervical spinal cord and nerve roots. Lateral-type OPLL might contribute to the development of C5 palsy due to the imbalanced stress and tension on the nerve roots after laminoplasty.

Postnatal maturation of mouse medullo-spinal cerebrospinal fluid-contacting neurons.

The central canal along the spinal cord (SC.) and medulla is characterized by the presence of a specific population of neurons that contacts the cerebrospinal fluid (CSF). These medullo-spinal CSF-contacting neurons (CSF-cNs) are identified by the selective expression of the polycystin kidney disease 2-like 1 ionic channel (PKD2L1 or polycystin-L). In adult, they have been shown to express doublecortin (DCX) and Nkx6.1, two markers of juvenile neurons along with the neuron-specific nuclear protein (NeuN) typically expressed in mature neurons. They were therefore suggested to remain in a rather incomplete maturation state. The aim of this study was to assess whether such juvenile state is stable in postnatal animals or whether CSF-cNs may reach maturity at older stages than neurons in the parenchyma. We show, in the cervical SC. and the brainstem that, in relation to age, CSF-cN density declines and that their cell bodies become more distant from the cc, except in its ventral part. Moreover, in adults (from 1month) by comparison with neonatal mice, we show that CSF-cNs have evolved to a more mature state, as indicated by the increase in the percentage of cells positive for NeuN and of its level of expression. In parallel, CSF-cNs exhibit, in adult, lower DCX immunoreactivity and do not express PSA-NCAM and TUC4, two neurogenic markers. Nevertheless, CSF-cNs still share in adult characteristics of juvenile neurons such as the presence of phospho-CREB and DCX while NeuN expression remained low. This phenotype persists in 12-month-old animals. Thus, despite a pursuit of neuronal maturation during the postnatal period, CSF-cNs retain a durable low differentiated state.