Spinal Projections Research Articles

Parallel ascending spinal pathways for affective touch and pain.

SummaryThe anterolateral pathway consists of ascending spinal tracts that convey pain, temperature and touch information from the spinal cord to the brain1–4. Projection neurons (PNs) of the anterolateral pathway are attractive therapeutic targets for pain treatment because nociceptive signals emanating from the periphery channel through these spinal PNs en route to the brain. However, the organizational logic of the anterolateral pathway remains elusive. Here, we show that two PN populations that express structurally related GPCRs, TACR1 and GPR83, form parallel ascending circuit modules that cooperate to convey tactile, thermal and noxious cutaneous signals from the spinal cord to the lateral parabrachial nucleus of the pons (PBNL). Axons of Tacr1- and Gpr83-expressing spinoparabrachial (SPB) neurons innervate distinct sets of PBNL subnuclei, and strong optogenetic stimulation of their axon terminals induces distinct escape behaviors and autonomic responses. Moreover, Gpr83-expressing SPB neurons are highly sensitive to cutaneous mechanical stimuli and receive strong synaptic inputs from both high- and low-threshold primary mechanosensory neurons. Remarkably, the valence associated with activation of Gpr83-expressing SPB neurons is either positive or negative depending on stimulus intensity. These findings reveal anatomically, physiologically, and functionally distinct SPB tract subdivisions that underlie affective aspects of touch and pain.

Chronic Pain Releases Parabrachial Activity from Central Amygdala Inhibition.

Chronic pain is a complex neurologic condition that involves both sensory and affective components. A crucial integrator of pain-related sensation and affect may be a set of neurons within the parabrachial nucleus. This area receives nociceptive inputs from spinal projection neurons and transmits

The Neurokinin-1 Receptor is Expressed with Gastrin-Releasing Peptide Receptor in Spinal Interneurons and Modulates Itch.

The neurokinin-1 receptor (NK1R; encoded by Tacr1) is expressed in spinal dorsal horn neurons and has been suggested to mediate itch in rodents. However, previous studies relied heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their function in spinal itch circuitry. To address this limitation, we leveraged a newly developed Tacr1CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1CreER spinal neurons increases itch behavior in male and female mice, whereas pharmacological inhibition of spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization (FISH) to characterize the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn (DDH), as well as the lateral spinal nucleus (LSN), of mouse and human spinal cord. Retrograde labeling studies in mice from the parabrachial nucleus (PBN) show that less than 20% of superficial Tacr1CreER dorsal horn neurons are spinal projection neurons, and thus the majority of Tacr1CreER are local interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca2+ imaging of the mouse spinal cord to establish that NK1R and the gastrin-releasing peptide receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn (SDH) neurons. These findings are the first to suggest a role for NK1R interneurons in itch and extend our understanding of the complexities of spinal itch circuitry.SIGNIFICANCE STATEMENT The spinal cord is a critical hub for processing somatosensory input, yet which spinal neurons process itch input and how itch signals are encoded within the spinal cord is not fully understood. We demonstrate neurokinin-1 receptor (NK1R) spinal neurons mediate itch behavior in mice and that the majority of NK1R spinal neurons are local interneurons. These NK1R neurons comprise a subset of gastrin-releasing peptide receptor (GRPR) interneurons and are thus positioned at the center of spinal itch transmission. We show NK1R mRNA expression in human spinal cord, underscoring the translational relevance of our findings in mice. This work is the first to suggest a role for NK1R interneurons in itch and extends our understanding of the complexities of spinal itch circuitry.

The Parabrachial Nucleus Directly Channels Spinal Nociceptive Signals to the Intralaminar Thalamic Nuclei, but Not the Amygdala

The parabrachial nucleus (PBN) is one of the major targets of spinal projection neurons and plays important roles in pain. However, the architecture of the spinoparabrachial pathway underlying its functional role in nociceptive information processing remains elusive. Here, we report that the PBN directly relays nociceptive signals from the spinal cord to the intralaminar thalamic nuclei (ILN). We demonstrate that the spinal cord connects with the PBN in a bilateral manner and that the ipsilateral spinoparabrachial pathway is critical for nocifensive behavior. We identify Tacr1-expressing neurons as the major neuronal subtype in the PBN that receives direct spinal input and show that these neurons are critical for processing nociceptive information. Furthermore, PBN neurons receiving spinal input form functional monosynaptic excitatory connections with neurons in the ILN, but not the amygdala. Together, our results delineate the neural circuit underlying nocifensive behavior, providing crucial insight into the circuit mechanism underlying nociceptive information processing.

Divergent Neural Pathways Emanating from the Lateral Parabrachial Nucleus Mediate Distinct Components of the Pain Response

The lateral parabrachial nucleus (lPBN) is a major target of spinal projection neurons conveying nociceptive input into supraspinal structures. However, the functional role of distinct lPBN efferents in diverse nocifensive responses have remained largely uncharacterized. Here we show that that the lPBN is required for escape behaviors and aversive learning to noxious stimulation. In addition, we find that two populations of efferent neurons from different regions of the lPBN collateralize to distinct targets. Activation of efferent projections to the ventromedial hypothalamus (VMH) or lateral periaqueductal gray (lPAG) drives escape behaviors, whereas activation of lPBN efferents to the bed nucleus stria terminalis (BNST) or central amygdala (CEA) generates an aversive memory. Finally, we provide evidence that dynorphin-expressing neurons, which span cytoarchitecturally distinct domains of the lPBN, are required for aversive learning.

Connection Input Mapping and 3D Reconstruction of the Brainstem and Spinal Cord Projections to the CSF-Contacting Nucleus.

ObjectiveTo investigate whether the CSF-contacting nucleus receives brainstem and spinal cord projections and to understand the functional significance of these connections.MethodsThe retrograde tracer cholera toxin B subunit (CB) was injected into the CSF-contacting nucleus in Sprague-Dawley rats according the previously reported stereotaxic coordinates. After 7–10 days, these rats were perfused and their brainstem and spinal cord were sliced (thickness, 40 μm) using a freezing microtome. All the sections were subjected to CB immunofluorescence staining. The distribution of CB-positive neuron in different brainstem and spinal cord areas was observed under fluorescence microscope.ResultsThe retrograde labeled CB-positive neurons were found in the midbrain, pons, medulla oblongata, and spinal cord. Four functional areas including one hundred and twelve sub-regions have projections to the CSF-contacting nucleus. However, the density of CB-positive neuron distribution ranged from sparse to dense.ConclusionBased on the connectivity patterns of the CSF-contacting nucleus receives anatomical inputs from the brainstem and spinal cord, we preliminarily conclude and summarize that the CSF-contacting nucleus participates in pain, visceral activity, sleep and arousal, emotion, and drug addiction. The present study firstly illustrates the broad projections of the CSF-contacting nucleus from the brainstem and spinal cord, which implies the complicated functions of the nucleus especially for the unique roles of coordination in neural and body fluids regulation.

Direct comparison of cervical and high thoracic spinal cord injury reveals distinct autonomic and cardiovascular consequences.

A wide range of spinal cord levels (cervical 8-thoracic 6) project to the stellate ganglia (which provides >90% of sympathetic supply to the heart), with a peak at the thoracic 2 (T2) level. We hypothesize that despite the proximity of the lesions, high thoracic spinal cord injuries (i.e., T2-3 SCI) do not closely mimic the hemodynamic responses recorded with cervical SCI (i.e., C6-7 SCI). To test this hypothesis, rats were instrumented with an intra-arterial telemetry device (Data Sciences International PA-C40) for recording arterial pressure, heart rate, and locomotor activity as well as a catheter within the intraperitoneal space. After recovery, rats were subjected to complete C6-7 spinal cord transection (n = 8), sham transection (n = 4), or T2-3 spinal cord transection (n = 7). After the spinal cord transection or sham transection, arterial pressure, heart rate, and activity counts were recorded in conscious animals, in a thermoneutral environment, for 20 s every minute, 24 h/day for 12 consecutive weeks. After 12 wk, chronic reflex- and stress-induced cardiovascular and hormonal responses were compared in all groups. C6-7 rats had hypotension, bradycardia, and reduced physical activity. In contrast, T2-3 rats were tachycardic. C6-7 rats compared with T2-3 and spinal intact rats also had reduced cardiac sympathetic tonus, reduced reflex- and stress induced cardiovascular responses, and reduced sympathetic support of blood pressure as well as enhanced reliance on angiotensin to maintain arterial blood pressure. Thus injuries above and below the peak level (T2) of spinal cord projections to the stellate ganglia have remarkably different outcomes.NEW & NOTEWORTHY Twelve consecutive weeks of resting hemodynamic data as well as chronic reflex- and stress-induced cardiovascular, autonomic, and hormonal responses were compared in spinal intact and C6-7 and T2-3 spinal cord-transected rats. C6-7 rats compared with T2-3 and spinal intact rats had reduced cardiac sympathetic tonus, reduced reflex- and stress-induced cardiovascular responses, and reduced sympathetic support of blood pressure as well as enhanced reliance on angiotensin to maintain arterial blood pressure. Thus injuries above and below the peak level (T2) of spinal cord projections to the stellate ganglia have remarkably different outcomes.

Abnormal Pyramidal Decussation and Bilateral Projection of the Corticospinal Tract Axons in Mice Lacking the Heparan Sulfate Endosulfatases, Sulf1 and Sulf2.

The corticospinal tract (CST) plays an important role in controlling voluntary movement. Because the CST has a long trajectory throughout the brain toward the spinal cord, many axon guidance molecules are required to navigate the axons correctly during development. Previously, we found that double-knockout (DKO) mouse embryos lacking the heparan sulfate endosulfatases, Sulf1 and Sulf2, showed axon guidance defects of the CST owing to the abnormal accumulation of Slit2 protein on the brain surface. However, postnatal development of the CST, especially the pyramidal decussation and spinal cord projection, could not be assessed because DKO mice on a C57BL/6 background died soon after birth. We recently found that Sulf1/2 DKO mice on a mixed C57BL/6 and CD-1/ICR background can survive into adulthood and therefore investigated the anatomy and function of the CST in the adult DKO mice. In Sulf1/2 DKO mice, abnormal dorsal deviation of the CST fibers on the midbrain surface persisted after maturation of the CST. At the pyramidal decussation, some CST fibers located near the midline crossed the midline, whereas others located more laterally extended ipsilaterally. In the spinal cord, the crossed CST fibers descended in the dorsal funiculus on the contralateral side and entered the contralateral gray matter normally, whereas the uncrossed fibers descended in the lateral funiculus on the ipsilateral side and entered the ipsilateral gray matter. As a result, the CST fibers that originated from 1 side of the brain projected bilaterally in the DKO spinal cord. Consistently, microstimulation of 1 side of the motor cortex evoked electromyogram responses only in the contralateral forelimb muscles of the wild-type mice, whereas the same stimulation evoked bilateral responses in the DKO mice. The functional consequences of the CST defects in the Sulf1/2 DKO mice were examined using the grid-walking, staircase, and single pellet-reaching tests, which have been used to evaluate motor function in mice. Compared with the wild-type mice, the Sulf1/2 DKO mice showed impaired performance in these tests, indicating deficits in motor function. These findings suggest that disruption of Sulf1/2 genes leads to both anatomical and functional defects of the CST.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

SummaryVolatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

Refinement of the dopaminergic system of anuran amphibians based on connectivity with habenula, basal ganglia, limbic system, pallium, and spinal cord.

Whereas our understanding of the dopaminergic system in mammals allows for a distinction between ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), no clear evidence for separate structures in anamniotes has been presented to date. To broaden the insight into the organization and regulation of neuromodulatory systems in anuran amphibians, tracing and immunohistochemical investigations were performed in the Oriental fire-bellied toad, Bombina orientalis. Topographically organized catecholaminergic "nigrostriatal," "mesolimbic," "mesocortical," and spinal cord projections arising from the posterior tubercle and mesencephalic tegmentum were identified. We compared these results with published data from lampreys, chondrichthyes, teleosts, amphibians, reptiles, birds, and mammals. Based on the pattern of organization, as well as the differential innervation by the habenular nuclei, domains gradually comparable to the mammalian paranigral VTA, ventral tier of the SNc, interfascicular nucleus of the VTA, and supramamillary/retromamillary area were identified. Additionally, we could demonstrate topographic separate populations of habenula neurons projecting via a direct excitatory or indirect GABAergic pathway onto the catecholaminergic VTA/SNc homologs and serotonergic raphe nuclei. The indirect GABAergic habenula pathway derives from neurons in the superficial mamillary area, which in terms of its connectivity and chemoarchitecture resembles the mammalian rostromedial tegmental nucleus. These results demonstrate a much more elaborate interconnection principle of the anuran dopaminergic system than previously assumed. Based on the data presented it seems that most features of the dopaminergic system of amniotes had already evolved when the amphibian line of evolution diverged from that leading up to mammals, reptiles, and birds.

The mammalian spinal commissural system: properties and functions.

Commissural systems are essential components of motor circuits that coordinate left-right activity of the skeletomuscular system. Commissural systems are found at many levels of the neuraxis including the cortex, brainstem, and spinal cord. In this review we will discuss aspects of the mammalian spinal commissural system. We will focus on commissural interneurons, which project from one side of the cord to the other and form axonal terminations that are confined to the cord itself. Commissural interneurons form heterogeneous populations and influence a variety of spinal circuits. They can be defined according to a variety of criteria including, location in the spinal gray matter, axonal projections and targets, neurotransmitter phenotype, activation properties, and embryological origin. At present, we do not have a comprehensive classification of these cells, but it is clear that cells located within different areas of the gray matter have characteristic properties and make particular contributions to motor circuits. The contribution of commissural interneurons to locomotor function and posture is well established and briefly discussed. However, their role in other goal-orientated behaviors such as grasping, reaching, and bimanual tasks is less clear. This is partly because we only have limited information about the organization and functional properties of commissural interneurons in the cervical spinal cord of primates, including humans. In this review we shall discuss these various issues. First, we will consider the properties of commissural interneurons and subsequently examine what is known about their functions. We then discuss how they may contribute to restoration of function following spinal injury and stroke.

Selective-cold output through a distinct subset of lamina I spinoparabrachial neurons.

Spinal projection neurons are a major pathway through which somatic stimuli are conveyed to the brain. However, the manner in which this information is coded is poorly understood. Here, we report the identification of a modality-selective spinoparabrachial (SPB) neuron subtype with unique properties. Specifically, we find that cold-selective SPB neurons are differentiated by selective afferent input, reduced sensitivity to substance P, distinct physiological properties, small soma size, and low basal drive. In addition, optogenetic experiments reveal that cold-selective SPB neurons do not receive input from Nos1 inhibitory interneurons and, compared with other SPB neurons, show significantly smaller inhibitory postsynaptic currents upon activation of Pdyn inhibitory interneurons. Together, these data suggest that cold output from the spinal cord to the parabrachial nucleus is mediated by a specific cell type with distinct properties.

Neonatal Injury Alters Sensory Input and Synaptic Plasticity in GABAergic Interneurons of the Adult Mouse Dorsal Horn.

Neonatal tissue injury disrupts the balance between primary afferent-evoked excitation and inhibition onto adult spinal projection neurons. However, whether this reflects cell-type-specific alterations at synapses onto ascending projection neurons, or rather is indicative of global changes in synaptic signaling across the mature superficial dorsal horn (SDH), remains unknown. Therefore the present study investigated the effects of neonatal surgical injury on primary afferent synaptic input to adult mouse SDH interneurons using in vitro patch-clamp techniques. Hindpaw incision at postnatal day (P)3 significantly diminished total primary afferent-evoked glutamatergic drive to adult Gad67-GFP and non-GFP neurons, and reduced their firing in response to sensory input, in both males and females. Early tissue damage also shaped the relative prevalence of monosynaptic A- versus C-fiber-mediated input to mature GABAergic neurons, with an increased prevalence of Aβ- and Aδ-fiber input observed in neonatally-incised mice compared with naive littermate controls. Paired presynaptic and postsynaptic stimulation at an interval that exclusively produced spike timing-dependent long-term potentiation (t-LTP) in projection neurons predominantly evoked NMDAR-dependent long-term depression in naive Gad67-GFP interneurons. Meanwhile, P3 tissue damage enhanced the likelihood of t-LTP generation at sensory synapses onto the mature GABAergic population, and increased the contribution of Ca2+-permeable AMPARs to the overall glutamatergic response. Collectively, the results indicate that neonatal injury suppresses sensory drive to multiple subpopulations of interneurons in the adult SDH, which likely represents one mechanism contributing to reduced feedforward inhibition of ascending projection neurons, and the priming of developing pain pathways, following early life trauma.SIGNIFICANCE STATEMENT Mounting clinical and preclinical evidence suggests that neonatal tissue damage can result in long-term changes in nociceptive processing within the CNS. Although recent work has demonstrated that early life injury weakens the ability of sensory afferents to evoke feedforward inhibition of adult spinal projection neurons, the underlying circuit mechanisms remain poorly understood. Here we demonstrate that neonatal surgical injury leads to persistent deficits in primary afferent drive to both GABAergic and presumed glutamatergic neurons in the mature superficial dorsal horn (SDH), and modifies activity-dependent plasticity at sensory synapses onto the GABAergic population. The functional denervation of spinal interneurons within the mature SDH may contribute to the "priming" of developing pain pathways following early life injury.

A Subfornical Organ→Paraventricular Nucleus Neuronal Network Contributes to Non‐Alcoholic Fatty Liver Disease via Hepatic Sympathetic Outflow

Non‐alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, affects 1 in 3 adults and leads to an increased risk for metabolic and cardiovascular diseases. Our recent work indicates that obesity‐induced NAFLD is mediated by elevations in hepatic sympathetic nerve activity. However, the neural pathways that contribute to hepatic sympathetic overactivity and subsequent NAFLD development remain unknown. The subfornical organ (SFO) ‐ a forebrain circumventricular region that lies outside the blood brain barrier ‐ senses, integrates and responds to circulating stimuli. Anatomical and electrophysiological data support dense, excitatory projections from the SFO to the paraventricular nucleus of the hypothalamus (SFO→PVN), an integrative nucleus with direct hepatic spinal projections. Taken together, we hypothesized that an SFO→PVN excitatory neuronal network causes hepatic steatosis via elevations in liver sympathetic outflow. An intersectional viral strategy was used in which a retrograde transported canine adenovirus was targeted to the PVN to express Cre‐recombinase in SFO→PVN neurons (CAV2‐Cre‐GFP). This was combined with SFO‐targeted delivery of a Cre‐inducible designer receptors engineered against designer drugs (DREADDs) excitatory construct (AAV2‐DIO‐hM3Gq‐mCherry). With this approach, the pharmacological ligand clozapine‐N‐oxide (CNO; 3 mg/kg i.p.) was administered once daily over 6 days to activate SFO→PVN neurons (n=4). Short‐term activation of SFO→PVN neurons resulted in hepatic steatosis (2.6±0.02 vs 2.9±0.02 density*107, saline vs CNO, p<0.05) that was paralleled by an elevation in liver tyrosine hydroxylase protein (1.8±0.4 CNO fold saline, p<0.05), the rate‐limiting enzyme for catecholamine synthesis within postganglionic nerve terminals. Based on this, we combined the above viral strategy with selective hepatic denervation (phenol application) or sham surgery (n=5–6). Oil Red O staining (Figure 1) demonstrated that hepatic denervation prevented liver lipid accumulation in response to 6‐day activation of SFO→PVN neurons (1.9±0.01 vs 1.7±0.01 density*107, CNO sham vs CNO denervation, p<0.05). Importantly, this occurred independent of changes in body weight and food intake. Hepatic denervation was confirmed by a reduction in liver tyrosine hydroxlase protein expression (0.3±0.1 vs 1.8±0.3 vs 0.4±0.1 fold saline sham, CNO sham vs saline denervation vs CNO denervation, all p<0.05). Lastly, male C57Bl/6 mice that were fed a high fat diet (10 wks) underwent intersectional viral targeting for expression of an inhibitory DREADDs construct (AAV2‐DIO‐hM4Gi‐mCherry) in SFO→PVN neurons. Remarkably, acute inhibition of SFO→PVN neurons in obese mice resulted in an approximate 45% reduction in hepatic steatosis (Figure 2, n=4/group). Collectively, these findings indicate that activation of SFO→PVN neurons causes liver triglyceride deposition via elevations in hepatic sympathetic outflow. Furthermore, in the context of obesity, inhibition of this forebrain‐hypothalamic‐autonomic circuit results in a marked reduction in NAFLD.Support or Funding InformationR01DK117007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(226) Gastrin-Releasing Peptide Defines an Itch-Specific Circuit in the Spinal Dorsal Horn

Recent studies have indicated that the spinal activity of gastrin-releasing peptide (GRP) and its receptor, GRPR, selectively modulate itching behaviors over other somatosensations including pain behaviors. Despite behavioral selectivity, how GRP acts within the spinal cord to selectively convey itch to the brain is not understood. Utilizing a novel ex vivo somatosensory preparation, retrograde labeling of spinal projection neurons, and multiphoton Ca2+ imaging, the activity of excitatory interneurons and projection neurons in the ex vivo spinal cord can be recorded in response to natural stimulation of the skin and pharmacologic modulation of GRP signaling. Using this technique to visualize activity, we found that GRP activates a primary population of neurons at the spinal dorsal horn which subsequently drives activity in a larger secondary population of interneurons. The activity of this circuit also drives output from the spinal dorsal horn through activation of local projection neurons. These experiments begin to define a network of neurons that may be involved in itch-selective signaling.

(230) The Role of nNOS Positive Interneurons in Spinal Somatosensory Circuits

Somatosensory information, encoding pain, touch, itch, heat and cold all enter the central nervous system at the spinal cord dorsal horn. Here this information is modulated through both descending control and local microcircuitry before being relayed to higher brain centres for perception of the stimulus. In this study, we use a semi intact, ex vivo preparation to investigate the role of a population of dorsal horn interneurons that express neuronal nitric oxide synthase (nNOS) in local spinal microcircuitry. In this preparation we dissect (in continuum) spinal cord, together with L2 and L3 roots, dorsal root ganglia, saphenous nerve and hind limb skin. This allows natural stimulation of the skin while performing whole cell patch clamp recordings from identified dorsal horn neurons. nNOScreER mice were crossed with either Ai9 or Ai32 reporter animals and given tamoxifen (0.4mg/kg, i.p) at day P14. Electrophysiology experiments were performed 3 weeks later. In some cases, DiI was injected to the parabrachial nucleus 4 days before experiments to retrogradely label spinal projection neurons. We found that nNOS positive interneurons receive input from both innocuous and noxious mechanical stimulation, but not a scratch stimulus. Further, optogenetic activation of these interneurons results in action potential discharge in identified projection neurons, suggesting an ability to send sensory information to higher brain areas. Using this ex vivo preparation will allow us to characterise spinal somatosensory circuits both under normal conditions (current study) and both acute and chronic pain states. This will help us understand how spinal microcircuitry is disrupted following injury.

(223) Anatomical Organization and Functional Contributions of NK1R Spinal Projection Neurons in Pain and Itch

Dissecting the spinal circuitry of pain and itch has been limited by a lack of genetic tools to label discrete subpopulations of spinal neurons. In particular, markers for spinal projection neurons are lacking, and thus the contributions of spinal projection neurons to somatosensation are largely unknown. Currently the only known marker for spinal projection neurons is neurokinin-1 receptor (NK1R), which captures the majority of spinal projection neurons. While neurotoxic ablation studies have provided evidence demonstrating the importance of NK1R spinal projection neurons to itch and hyperalgesia, interpretation of these studies is limited by ambiguities in the time course, specificity, and extent of neuronal death. Here we describe our recently developed NK1R-CreER mouse line as a tool to dissect the spinal circuitry of somatosensation. Using this novel genetic tool, we visualize the outputs of NK1R spinal projection neurons, manipulate their activity and determine their contribution to pain and itch behaviors, and identify new molecular markers using recently published single cell RNA-sequencing of spinal cord dorsal horn neuron populations. These studies shed light on the anatomical organization and functional contributions of NK1R spinal projection neurons to pain and itch. Supported by T32NS086749 and F32NS110155 to TDS and R01NS096705 to SER.

A hierarchical classification of adolescent idiopathic scoliosis: Identifying the distinguishing features in 3D spinal deformities.

This study aimed to identify the differentiating parameters of the spinal curves’ 2D projections through a hierarchical classification of the 3D spinal curve in adolescent idiopathic scoliosis (AIS). A total number of 103 right thoracic left lumbar pre-operative AIS patients were included retrospectively and consecutively. A total number of 20 non-scoliotic adolescents were included as the control group. All patients had biplanar X-rays and 3D reconstructions of the spine. The 3D spinal curve was calculated by interpolating the center of vertebrae and was isotropically normalized. A hierarchical classification of the normalized spinal curves was developed to group the patients based on the similarity of their 3D spinal curve. The spinal curves’ 2D projections and clinical spinal measurements in the three anatomical planes were then statistically compared between these groups and between the scoliotic subtypes and the non-scoliotic controls. A total of 5 patient groups of right thoracic left lumbar AIS patients were identified. The characteristics of the posterior-anterior and sagittal views of the spines were: Type 1: Normal sagittal profile and S shape axial view. T1 is leveled or tilted to the right in the posterior view. Type 2: Hypokyphotic and a V shape axial view. T1 is tilted to the left in the posterior view. Type 3: Hypokyphotic (only T5-T10) and frontal imbalance, S shape axial view. T1 is leveled or tilted to the right, and 3 frontal curves. Type 4: Flat sagittal profile (T1-L2), slight frontal imbalance with a V shape axial view, T1 tilted to the left. Type 5: flat sagittal profile and forward trunk shift with a proximal kyphosis and S shape axial view. T1 is leveled or tilted to the right. In conclusion, a hierarchical classification of the 3D scoliotic spine allowed identifying various distinguishing features of the spinal curves in patients with a right thoracic curve in an orderly fashion. The subtypes’ characteristics resulting from this 3D classification can be identified from the pairs of the frontal and sagittal spinal curves i.e. X-rays in right thoracic AIS patients.

The greater occipital nerve and its spinal and brainstem afferent projections: A stereological and tract-tracing study in the rat.

The neuromodulation of the greater occipital nerve (GON) has proved effective to treat chronic refractory neurovascular headaches, in particular migraine and cluster headache. Moreover, animal studies have shown convergence of cervical and trigeminal afferents on the same territories of the upper cervical and lower medullary dorsal horn (DH), the so-called trigeminocervical complex (TCC), and recent studies in rat models of migraine and craniofacial neuropathy have shown that GON block or stimulation alter nociceptive processing in TCC. The present study examines in detail the anatomy of GON and its central projections in the rat applying different tracers to the nerve and quantifying its ultrastructure, the ganglion neurons subserving GON, and their innervation territories in the spinal cord and brainstem. With considerable intersubject variability in size, GON contains on average 900 myelinated and 3,300 unmyelinated axons, more than 90% of which emerge from C2 ganglion neurons. Unmyelinated afferents from GON innervates exclusively laminae I-II of the lateral DH, mostly extending along segments C2-3 . Myelinated fibers distribute mainly in laminae I and III-V of the lateral DH between C1 and C6 and, with different terminal patterns, in medial parts of the DH at upper cervical segments, and ventrolateral rostral cuneate, paratrigeminal, and marginal part of the spinal caudal and interpolar nuclei. Sparse projections also appear in other locations nearby. These findings will help to better understand the bases of sensory convergence on spinomedullary systems, a critical pathophysiological factor for pain referral and spread in severe painful craniofacial disorders.

Organization of the Thermal Grill Illusion by Spinal Segments.

ObjectiveA common symptom of neuropathy is the misperception of heat and pain from cold stimuli. Similar cold allodynic sensations can be experimentally induced using the thermal grill illusion (TGI) in humans. It is currently unclear whether this interaction between thermosensory and nociceptive signals depends on spinal or supraspinal integration mechanisms. To address this issue, we developed a noninvasive protocol to assess thermosensory integration across spinal segments.MethodsWe leveraged anatomical knowledge regarding dermatomes and their spinal projections to investigate potential contributions of spinal integration to the TGI. We simultaneously stimulated a pair of skin locations on the arm or lower back using 1 cold (∼20°C) and 1 warm thermode (∼40°C). The 2 thermodes were always separated by a fixed physical distance on the skin, but elicited neural activity across a varying number of spinal segments, depending on which dermatomal boundaries the 2 stimuli spanned.ResultsParticipants consistently overestimated the actual cold temperature on the skin during combined cold and warm stimulation, confirming the TGI effect. The TGI was present when cold and warm stimuli were delivered within the same dermatome, or across dermatomes corresponding to adjacent spinal segments. In striking contrast, no TGI effect was found when cold and warm stimuli projected to nonadjacent spinal segments.InterpretationThese results demonstrate that the strength of the illusion is modulated by the segmental distance between cold and warm afferents. This suggests that both temperature perception and thermal–nociceptive interactions depend upon low‐level convergence mechanisms operating within a single spinal segment and its immediate neighbors. Ann Neurol 2018;84:463–472