The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity.

Growth of intact axons of noninjured neurons, often termed collateral sprouting, contributes to both adaptive and pathological plasticity in the adult nervous system, but the intracellular factors controlling this growth are largely unknown. An automated functional assay of genes regulated in sensory neurons from the rat in vivo spared dermatome model of collateral sprouting identified the adaptor protein CD2-associated protein (CD2AP; human CMS) as a positive regulator of axon growth. In non-neuronal cells, CD2AP, like other adaptor proteins, functions to selectively control the spatial/temporal assembly of multiprotein complexes that transmit intracellular signals. Although CD2AP polymorphisms are associated with increased risk of late-onset Alzheimer's disease, its role in axon growth is unknown. Assessments of neurite arbor structure in vitro revealed CD2AP overexpression, and siRNA-mediated knockdown, modulated (1) neurite length, (2) neurite complexity, and (3) growth cone filopodia number, in accordance with CD2AP expression levels. We show, for the first time, that CD2AP forms a novel multiprotein complex with the NGF receptor TrkA and the PI3K regulatory subunit p85, with the degree of TrkA:p85 association positively regulated by CD2AP levels. CD2AP also regulates NGF signaling through AKT, but not ERK, and regulates long-range signaling though TrkA(+)/RAB5(+) signaling endosomes. CD2AP mRNA and protein levels were increased in neurons during collateral sprouting but decreased following injury, suggesting that, although typically considered together, these two adult axonal growth processes are fundamentally different. These data position CD2AP as a major intracellular signaling molecule coordinating NGF signaling to regulate collateral sprouting and structural plasticity of intact adult axons. SIGNIFICANCE STATEMENT: Growth of noninjured axons in the adult nervous system contributes to adaptive and maladaptive plasticity, and dysfunction of this process may contribute to neurologic pathologies. Functional screening of genes regulated during growth of noninjured axons revealed CD2AP as a positive regulator of axon outgrowth. A novel association of CD2AP with TrkA and p85 suggests a distinct intracellular signaling pathway regulating growth of noninjured axons. This may also represent a novel mechanism of generating specificity in multifunctional NGF signaling. Divergent regulation of CD2AP in different axon growth conditions suggests that separate mechanisms exist for different modes of axon growth. CD2AP is the first signaling molecule associated with adult sensory axonal collateral sprouting, and this association may offer new insights for NGF/TrkA-related Alzheimer's disease mechanisms.

Neurotrophic factors in spinal cord injury.

A major challenge in repairing the injured spinal cord is to assure survival of damaged cells and to encourage regrowth of severed axons. Because neurotrophins are known to affect these processes during development, many experimental approaches to improving function of the injured spinal cord have made use of these agents, particularly Brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3). More recently, neurotrophins have also been shown to affect the physiology of cells and synapses in the spinal cord. The effect of neurotrophins on circuit performance adds an important dimension to their consideration as agents for repairing the injured spinal cord. In this chapter we discuss the role of neurotrophins in promoting recovery after spinal cord injury from both a structural and functional perspective.

Computational functions of neurons and circuits signaling injury: relationship to pain behavior.

The basic circuitry of the "pain pathway" mediating transmission of information from the periphery to the brain is well known, consisting of specialized sensory fibers known as nociceptors projecting to specific spinal cord neurons, which in turn project on to the thalamus and cerebral cortex. Here we survey some of the unique properties of these circuits, such as peripheral and central sensitization, and the segmental and descending modulatory control of synaptic transmission. We also review evidence indicating dissociation between nociceptor activity and behavioral indications of pain. Together, these considerations point to the need for a more quantitative approach to the nociceptive system, specifically the interactions at peripheral, spinal, and supraspinal levels as well as between them, to more fully understand how the activity in nociceptive neurons individually and collectively is related to the pain response.

Bilateral transient changes in thalamic nucleus ventroposterior lateralis after thoracic hemisection in the rat.

We made simultaneous bilateral recordings of unit activity in the nucleus ventroposterior lateralis (VPL) in intact rats and after acute and chronic left thoracic hemisection. We observed an immediate bilateral decline in multireceptive units, reflecting a loss of nociceptive input on the lesion side and a loss of low-threshold inputs contralaterally. Unit properties were restored to normal by 6 wk. Mean spontaneous discharge frequency remained unchanged in left VPL at all intervals. Right VPL displayed a substantial increase in spontaneous discharge frequency at 2 and 4 wk, returning to normal by 6 wk. Activity in left VPL driven by Pinch or Brush of the right limb was unchanged except for an immediate decrease in the response to Pinch, which was reversed by 2 wk despite persistent left hemisection. In right VPL, the response to Pinch or Brush of the left hindlimb was enhanced at 2 and 4 wk but returned to normal by 6 wk. Behaviorally, the same rats displayed increased sensitivity to mechanical stimulation of the left hindlimb, but, unlike VPL activity, there was no significant behavioral recovery. Bursting cells were also observed bilaterally in VPL, but this did not match the restriction of scratches to the hindlimb contralateral to the hemisection considered to be evidence for neuropathic pain. The novel findings include recovery of responsiveness to Pinch on the side ipsilateral to the hemisection despite the lack of spinothalamic input as well as failure for the thalamus contralateral to hemisection to maintain its elevated responsiveness.

Chondroitinase ABC combined with neurotrophin NT-3 secretion and NR2D expression promotes axonal plasticity and functional recovery in rats with lateral hemisection of the spinal cord.

Elevating spinal levels of neurotrophin NT-3 (NT3) while increasing expression of the NR2D subunit of the NMDA receptor using a HSV viral construct promotes formation of novel multisynaptic projections from lateral white matter (LWM) axons to motoneurons in neonates. However, this treatment is ineffective after postnatal day 10. Because chondroitinase ABC (ChABC) treatment restores plasticity in the adult CNS, we have added ChABC to this treatment and applied the combination to adult rats receiving a left lateral hemisection (Hx) at T8. All hemisected animals initially dragged the ipsilateral hindpaw and displayed abnormal gait. Rats treated with ChABC or NT3/HSV-NR2D recovered partial hindlimb locomotor function, but animals receiving combined therapy displayed the most improved body stability and interlimb coordination [Basso-Beattie-Bresnahan (BBB) locomotor scale and gait analysis]. Electrical stimulation of the left LWM at T6 did not evoke any synaptic response in ipsilateral L5 motoneurons of control hemisected animals, indicating interruption of the white matter. Only animals with the full combination treatment recovered consistent multisynaptic responses in these motoneurons indicating formation of a detour pathway around the Hx. These physiological findings were supported by the observation of increased branching of both cut and intact LWM axons into the gray matter near the injury. ChABC-treated animals displayed more sprouting than control animals and those receiving NT3/HSV-NR2D; animals receiving the combination of all three treatments showed the most sprouting. Our results indicate that therapies aimed at increasing plasticity, promoting axon growth and modulating synaptic function have synergistic effects and promote better functional recovery than if applied individually.

Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats.

We compared the effect of viral administration of brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT-3) on locomotor recovery in adult rats with complete thoracic (T10) spinal cord transection injuries, in order to determine the effect of chronic neurotrophin expression on spinal plasticity. At the time of injury, BDNF, NT-3 or green fluorescent protein (GFP) (control) was delivered to the lesion via adeno-associated virus (AAV) constructs. AAV-BDNF was significantly more effective than AAV-NT-3 in eliciting locomotion. In fact, AAV-BDNF-treated rats displayed plantar, weight-supported hindlimb stepping on a stationary platform, that is, without the assistance of a moving treadmill and without step training. Rats receiving AAV-NT-3 or AAV-GFP were incapable of hindlimb stepping during this task, despite provision of balance support. AAV-NT-3 treatment did promote the recovery of treadmill-assisted stepping, but this required continuous perineal stimulation. In addition, AAV-BDNF-treated rats were sensitized to noxious heat, whereas AAV-NT-3-treated and AAV-GFP-treated rats were not. Notably, AAV-BDNF-treated rats also developed hindlimb spasticity, detracting from its potential clinical applicability via the current viral delivery method. Intracellular recording from triceps surae motoneurons revealed that AAV-BDNF significantly reduced motoneuron rheobase, suggesting that AAV-BDNF promoted the recovery of over-ground stepping by enhancing neuronal excitability. Elevated nuclear c-Fos expression in interneurons located in the L2 intermediate zone after AAV-BDNF treatment indicated increased activation of interneurons in the vicinity of the locomotor central pattern generator. AAV-NT-3 treatment reduced motoneuron excitability, with little change in c-Fos expression. These results support the potential for BDNF delivery at the lesion site to reorganize locomotor circuits.

Role of chondroitin sulfate proteoglycans in axonal conduction in Mammalian spinal cord.

Chronic unilateral hemisection (HX) of the adult rat spinal cord diminishes conduction through intact fibers in the ventrolateral funiculus (VLF) contralateral to HX. This is associated with a partial loss of myelination from fibers in the VLF (Arvanian et al., 2009). Here, we again measured conduction through the VLF using electrical stimulation while recording the resulting volley and synaptic potentials in target motoneurons. We found that intraspinal injection of chondroitinase-ABC, known to digest chondroitin sulfate proteoglycans (CSPGs), prevented the decline of axonal conduction through intact VLF fibers across from chronic T10 HX. Chondroitinase treatment was also associated with behavior suggestive of an improvement of locomotor function after chronic HX. To further study the role of CSPGs in axonal conduction, we injected three purified CSPGs, NG2 and neurocan, which increase in the vicinity of a spinal injury, and aggrecan, which decreases, into the lateral column of the uninjured cord at T10 in separate experiments. Intraspinal injection of NG2 acutely depressed axonal conduction through the injected region in a dose-dependent manner. Similar injections of saline, aggrecan, or neurocan had no significant effect. Immunofluorescence staining experiments revealed the presence of endogenous and exogenous NG2 at some nodes of Ranvier. These results identify a novel acute action of CSPGs on axonal conduction in the spinal cord and suggest that antagonism of proteoglycans reverses or prevents the decline of axonal conduction, in addition to stimulating axonal growth.

Combined delivery of Nogo-A antibody, neurotrophin-3 and the NMDA-NR2d subunit establishes a functional 'detour' in the hemisected spinal cord.

To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by descending fibers after an intervening lateral thoracic (T10) hemisection (Hx), we treated adult rats with the following agents: (i) anti-Nogo-A antibodies to neutralize the growth-inhibitor Nogo-A; (ii) neurotrophin-3 (NT-3) via engineered fibroblasts to promote neuron survival and plasticity; and (iii) the NMDA-receptor 2d (NR2d) subunit via an HSV-1 amplicon vector to elevate NMDA receptor function by reversing the Mg(2+) block, thereby enhancing synaptic plasticity and promoting the effects of NT-3. Synaptic responses evoked by stimulation of the ventrolateral funiculus ipsilateral and rostral to the Hx were recorded intracellularly from ipsilateral lumbar motoneurons. In uninjured adult rats short-latency (1.7-ms) monosynaptic responses were observed. After Hx these monosynaptic responses were abolished. In the Nogo-Ab + NT-3 + NR2d group, long-latency (approximately 10 ms), probably polysynaptic, responses were recorded and these were not abolished by re-transection of the spinal cord through the Hx area. This suggests that these novel responses resulted from new connections established around the Hx. Anterograde anatomical tracing from the cervical grey matter ipsilateral to the Hx revealed increased numbers of axons re-crossing the midline below the lesion in the Nogo-Ab + NT-3 + NR2d group. The combined treatment resulted in slightly better motor function in the absence of adverse effects (e.g. pain). Together, these results suggest that the combination treatment with Nogo-Ab + NT-3 + NR2d can produce a functional 'detour' around the lesion in a laterally hemisected spinal cord. This novel combination treatment may help to improve function of the damaged spinal cord.

Antagonism of nerve growth factor-TrkA signaling and the relief of pain.

Nerve growth factor (NGF) was originally discovered as a neurotrophic factor essential for the survival of sensory and sympathetic neurons during development. However, in the adult NGF has been found to play an important role in nociceptor sensitization after tissue injury. The authors outline mechanisms by which NGF activation of its cognate receptor, tropomyosin-related kinase A receptor, regulates a host of ion channels, receptors, and signaling molecules to enhance acute and chronic pain. The authors also document that peripherally restricted antagonism of NGF-tropomyosin-related kinase A receptor signaling is effective for controlling human pain while appearing to maintain normal nociceptor function. Understanding whether there are any unexpected adverse events and how humans may change their behavior and use of the injured/degenerating tissue after significant pain relief without sedation will be required to fully appreciate the patient populations that may benefit from these therapies targeting NGF.

Intramuscular AAV delivery of NT-3 alters synaptic transmission to motoneurons in adult rats.

We examined whether elevating levels of neurotrophin-3 (NT-3) in the spinal cord and dorsal root ganglion (DRG) would alter connections made by muscle spindle afferent fibers on motoneurons. Adeno-associated virus (AAV) serotypes AAV1, AAV2 and AAV5, selected for their tropism profile, were engineered with the NT-3 gene and administered to the medial gastrocnemius muscle in adult rats. ELISA studies in muscle, DRG and spinal cord revealed that NT-3 concentration in all tissues peaked about 3 months after a single viral injection; after 6 months NT-3 concentration returned to normal values. Intracellular recording in triceps surae motoneurons revealed complex electrophysiological changes. Moderate elevation in cord NT-3 resulted in diminished segmental excitatory postsynaptic potential (EPSP) amplitude, perhaps as a result of the observed decrease in motoneuron input resistance. With further elevation in NT-3 expression, the decline in EPSP amplitude was reversed, indicating that NT-3 at higher concentration could increase EPSP amplitude. No correlation was observed between EPSP amplitude and NT-3 concentration in the DRG. Treatment with control viruses could elevate NT-3 levels minimally resulting in measurable electrophysiological effects, perhaps as a result of inflammation associated with injection. EPSPs elicited by stimulation of the ventrolateral funiculus underwent a consistent decline in amplitude independent of NT-3 level. These novel correlations between modified NT-3 expression and single-cell electrophysiological parameters indicate that intramuscular administration of AAV(NT-3) can exert long-lasting effects on synaptic transmission to motoneurons. This approach to neurotrophin delivery could be useful in modifying spinal function after injury.

Input-specific plasticity of N-methyl-D-aspartate receptor-mediated synaptic responses in neonatal rat motoneurons.

Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: the segmental dorsal root (DR) and the descending ventrolateral funiculus (VLF). To determine whether their N-methyl-d-aspartate (NMDA) receptors are independent, we used (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK-801), known to induce a use-dependent irreversible block of NMDA receptors (NMDARs). In the presence of MK-801 (in bath) and non-NMDA antagonists (in bath, to isolate NMDARs pharmacologically), we first stimulated the DR. After MK-801 blockade of DR synaptic input, the VLF was stimulated. Its response was found to be not significantly different from its control value, suggesting that the DR stimulus activated very few, if any, receptors also activated by VLF stimulation. Similar findings were obtained if the stimulation order was reversed. Both inputs also elicited a polysynaptic NMDAR-mediated response. Evoking the DR polysynaptic response in the presence of MK-801 eliminated the corresponding VLF response; the reverse did not occur. Surprisingly, when MK-801 was washed from the bath, both the DR and the VLF responses could recover, although the recovery of the DR monosynaptic and polysynaptic responses was reliably greater than those associated with the VLF. Recovery was prevented if extrasynaptic receptors were activated by bath-applied NMDA in the presence of MK-801, consistent with the possibility that recovery was due to movement of extrasynaptic receptors into parts of the membrane accessible to transmitter released by DR and VLF stimulation. These novel findings suggest that segmental glutamatergic inputs to motoneurons are more susceptible to plastic changes than those from central nervous system white matter inputs at this developmental stage.

Changes in motoneuron properties and synaptic inputs related to step training after spinal cord transection in rats.

Although recovery from spinal cord injury is generally meager, evidence suggests that step training can improve stepping performance, particularly after neonatal spinal injury. The location and nature of the changes in neural substrates underlying the behavioral improvements are not well understood. We examined the kinematics of stepping performance and cellular and synaptic electrophysiological parameters in ankle extensor motoneurons in nontrained and treadmill-trained rats, all receiving a complete spinal transection as neonates. For comparison, electrophysiological experiments included animals injured as young adults, which are far less responsive to training. Recovery of treadmill stepping was associated with significant changes in the cellular properties of motoneurons and their synaptic input from spinal white matter [ipsilateral ventrolateral funiculus (VLF)] and muscle spindle afferents. A strong correlation was found between the effectiveness of step training and the amplitude of both the action potential afterhyperpolarization and synaptic inputs to motoneurons (from peripheral nerve and VLF). These changes were absent if step training was unsuccessful, but other spinal projections, apparently inhibitory to step training, became evident. Greater plasticity of axonal projections after neonatal than after adult injury was suggested by anatomical demonstration of denser VLF projections to hindlimb motoneurons after neonatal injury. This finding confirmed electrophysiological measurements and provides a possible mechanism underlying the greater training susceptibility of animals injured as neonates. Thus, we have demonstrated an "age-at-injury"-related difference that may influence training effectiveness, that successful treadmill step training can alter electrophysiological parameters in the transected spinal cord, and that activation of different pathways may prevent functional improvement.

Spinal cord transection enhances afferent-evoked inhibition in lamina II neurons and abolishes BDNF-induced facilitation of their sensory input.

We previously reported that the pronociceptive neurotrophin brain-derived neurotrophic factor (BDNF) induces facilitation of C-fiber evoked EPSCs and NMDA currents in lamina II neurons of rats up to P40. Here, patch-clamp recording was used to study BDNF-induced modification of synaptic and NMDA-evoked responses in transverse spinal slices from lumbar (L2-L5) spinal cord of rats from P3 to P21 following complete spinal cord transection at P2. After transection injury at either T13/L1 or L6/S1, BDNF failed to facilitate synaptic AMPA-kainate currents or agonist-induced NMDA currents. The evoked synaptic currents were smaller in amplitude, often consisting of a biphasic (excitatory-inhibitory) response. The EPSC decayed more rapidly in neurons from transected cords than in those from uninjured cords. In transected cords most neurons responded to the GABA(A) receptor antagonist bicuculline with a significant increase in duration of the excitatory synaptic response. This could subsequently be blocked by D-APV, suggesting an NMDA-receptor mediated component. These findings suggest that following spinal cord transection, BDNF spinal actions are no longer predominantly pronociceptive. It is possible that a diminished availability of full-length TrkB and an enhanced expression of truncated TrkB receptors in the injured cord could play an important role in reducing the effect of BDNF following injury. These data are compared to those obtained after contusion, and it is concluded that the physiological changes after spinal injury differ according to nature of the injury.

Combined treatment with neurotrophin-3 and LSD facilitates behavioral recovery from double-hemisection spinal injury in neonatal rats.

We explored functional recovery in two spinal cord injury models following a novel combination treatment (NT-3 + LSD). One group of rats received a staggered double hemisection (DH) at postnatal day 2 (P2) of the left hemicord at T11 and the right hemicord at T12. Another group received complete transection (CT) at T11 on P2. A third group was sham operated. Each of these groups was also treated with the drug combination. Drugs were administered intrathecally above the lesion during surgery, and again s.c. at P4, P6, P8, and P10. Intracellular recording in an in vitro spinal cord preparation at P10-P12 in DH rats revealed weak polysynaptic connections to lumbar motoneurons through the injury region, but only in those receiving NT-3 + LSD; NT-3 or LSD alone had no effect. In behavioral experiments, the frequency of rearing in an open field and hindlimb kicks during swimming was assessed every 3-4 days from P9 to P58. Both CT and DH injury severely impaired rearing and hindlimb kicking during swimming. DH rats treated with NT-3 + LSD showed significantly more kicks during swimming than untreated DH or CT rats and treated CT rats beginning as early as P9 and lasting through the duration of testing. Rearing behavior was also improved by treatment but beginning only in the 3rd postnatal week, the time at which it normally develops. Rearing frequency reached sham control levels by P40. Our results suggest this combination treatment may be a promising new strategy for facilitating recovery from moderate spinal cord injury.

Chronic neurotrophin-3 strengthens synaptic connections to motoneurons in the neonatal rat.

We report that neurotrophin-3 (NT-3), delivered chronically via fibroblasts implanted intrathecally into neonatal rats, can facilitate synaptic transmission in the spinal cord. A small collagen plug containing NT-3-secreting fibroblasts was placed on the exposed dorsal surface of the spinal cord (L1) of 2-d-old rats; controls received beta-galactosidase-secreting fibroblasts. After 6 hr to 12 d of survival, synaptic potentials (EPSP) elicited by two synaptic inputs, L5 dorsal root and ventrolateral funiculus (VLF), were recorded intracellularly in L5 motoneurons in vitro. Preparations treated with NT-3 implants exhibited enhanced monosynaptic synaptic transmission from both inputs, which persisted over the entire testing period. Unlike acute enhancement of transmission by NT-3 (Arvanian and Mendell, 2001a), the chronic effect could occur at connections not normally eliciting an NMDA receptor-mediated response at the time of NT-3 exposure. Using susceptibility to blockade of the NMDA receptor by Mg2+ and APV, we confirmed that chronic treatment with NT-3 did not enhance NMDA receptor activity at these connections. Cords treated with chronic NT-3 also transiently displayed polysynaptic components activated by VLF that were blocked by the NMDA receptor antagonist APV. These novel NMDA receptor-mediated potentials may reflect changes in interneurons near the site of fibroblast implantation. We conclude that chronic NT-3 enhances the potency of segmental and descending projections via mechanisms different from those underlying acute changes.

Transcriptional changes in sensory ganglia associated with primary afferent axon collateral sprouting in spared dermatome model.

Primary afferent collateral sprouting is a process whereby non-injured primary afferent neurons respond to some stimulus and extend new branches from existing axons. Neurons of both the central and peripheral nervous systems undergo this process, which contributes to both adaptive and maladaptive plasticity (e.g., [1], [2], [3], [4], [5], [6], [7], [8], [9]). In the model used here (the "spared dermatome" model), the intact sensory neurons respond to the denervation of adjacent areas of skin by sprouting new axon branches into that adjacent denervated territory. Investigations of gene expression changes associated with collateral sprouting can provide a better understanding of the molecular mechanisms controlling this process. Consequently, it can be used to develop treatments to promote functional recovery for spinal cord injury and other similar conditions. This report includes raw gene expression data files from microarray experiments in order to study the gene regulation in spared sensory ganglia in the initiation (7 days) and maintenance (14 days) phases of the spared dermatome model relative to intact ("naïve") sensory ganglia. Data has been deposited into GEO (GSE72551).

Neurotrophin action on sensory neurons in adults: an extension of the neurotrophic hypothesis.

This brief review explores the action of neurotrophins on sensory neurons in adults. Neutralization of neurotrophins in adults does not cause sensory neurons to die as it does in prenatal animals. Thus they are not required as survival factors in adults. However, neurotrophins continue to play important roles in the postnatal development of sensory neurons. They also exert strong effects on the anatomy and physiology of these fibers after axotomy in adults. Here we review of the effects of NT-3 on spindle afferent fibers and NGF on nociceptive afferents and consider possible extension of the neurotrophic hypothesis to adults.

The many functions of nerve growth factor: multiple actions on nociceptors.

Nerve growth factor (NGF) initially interested neurobiologists because of its effects in the developing nervous system. It is now clear that NGF functions throughout the life of the animal with a wide repertoire of actions. In the sensory nervous system it primarily influences the structure and function of nociceptors. Here, we provide a brief review of these actions and raise the overriding biological questions of why these multiple actions occur and how they are carried out.

Constructing and deconstructing the gate theory of pain.

The gate theory of pain, published by Ronald Melzack and Patrick Wall in Science in 1965, was formulated to provide a mechanism for coding the nociceptive component of cutaneous sensory input. The theory dealt explicitly with the apparent conflict in the 1960s between the paucity of sensory neurons that responded selectively to intense stimuli and the well-established finding that stimulation of the small fibers in peripheral nerves is required for the stimulus to be described as painful. It incorporated recently discovered mechanisms of presynaptic control of synaptic transmission from large and small sensory afferents, which was suggested to "gate" incoming information depending on the balance between these inputs. Other important features included the convergence of small and large sensory inputs on spinal neurons that transmitted the sensory information to the forebrain as well as the ability of descending control pathways to affect the biasing established by the gate. The clarity of the model and its description gave this article immediate visibility, with numerous attempts made to test its various predictions. Although subsequent experiments and clinical findings have made clear that the model is not correct in detail, the general ideas put forth in the article and the experiments they prompted in both animals and patients have transformed our understanding of pain mechanisms.