FO C U S O N PA I N                                                                                                       ...
REVIEWS a Nociceptor specification         b Neuronal survival            c Axon growth                — NGN2 primarily fo...
FO C U S O N PA I N                                   Functional development of nociceptors                         termin...
REVIEWS                                   a                                                                 b             ...
FO C U S O N PA I N                                    tenfold higher than that of weanlings53,54. Until P10, the    relea...
REVIEWS                                                           Newborn                                                 ...
FO C U S O N PA I N                                     Box 1 | Development of excitatory synaptic transmission in the neo...
REVIEWS                                    Box 2 | Development of inhibitory transmission in the neonatal dorsal horn     ...
FO C U S O N PA I N                          a                                                                            ...
The development of nociceptive circuits
The development of nociceptive circuits
The development of nociceptive circuits
The development of nociceptive circuits
The development of nociceptive circuits
The development of nociceptive circuits
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The development of nociceptive circuits


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The development of nociceptive circuits

  1. 1. FO C U S O N PA I N REVIEWS THE DEVELOPMENT OF NOCICEPTIVE CIRCUITS Maria Fitzgerald Abstract | The study of pain development has come into its own. Reaping the rewards of years of developmental and molecular biology, it has now become possible to translate fundamental knowledge of signalling pathways and synaptic physiology into a better understanding of infant pain. Research has cast new light on the physiological and pharmacological processes that shape the newborn pain response, which will help us to understand early pain behaviour and to design better treatments. Furthermore, it has shown how developing pain circuitry depends on non-noxious sensory activity in the healthy newborn, and how early injury can permanently alter pain processing.TYROSINE KINASE RECEPTORS After years of neglect, the study of infant pain has nociceptive reflexes, emphasizing recent research on(Trk). Neurotrophic factors — emerged as one of the most interesting and important the postnatal developmental regulation of excitatorynerve growth factor (NGF), areas of pain research today. It provides a fascinating and inhibitory synaptic transmission in the developingneurotrophin 3 (NT3), NT4/5 insight into how immature neurons connect together dorsal horn. Finally, the role of sensory activity — fromand brain-derived neurotrophicfactor (BDNF) — act through a to analyse and process somatic events, and illustrates both non-noxious and excessive noxious inputsfamily of receptor proteins, the how the growing nervous system deals with events — in influencing the development of pain processingTrk receptors. TrkA is primarily that threaten its postnatal integrity and survival. It is reviewed and the implications for human infant painthe receptor for NGF, TrkB for also has the potential to provide us with a neurobio- discussed. Ultimately, we argue that we need to use thisBDNF and NT4/5, and TrkCfor NT3. logical basis for pain assessment and treatment in research to design better strategies for the relief of pain human infants. in infants and children. Newborn infants show strong pain behaviour, but the study of the development of nociceptive pathways Early specification of nociceptive neurons shows that their pain involves functional signalling Nociceptive neurons are specified early in develop- pathways that are not found in the mature nervous ment, long before they form contacts with their future system in healthy individuals. Recent research into peripheral or central targets. Sensory sublineages are the development of inhibitory and excitatory synaptic determined even before neural crest cells become transmission has provided new insights into the crucial committed to neuronal or glial fates1. Different classes changes that shape pain pathways in the first postnatal of sensory neurons in dorsal root ganglia (DRG) are weeks. generated in two waves: large-diameter TYROSINE KINASE + + This review focuses on the underlying organization RECEPTOR C- and B-expressing (TrkC and TrkB ) neu- and strengthening of nociceptive circuitry in the dorsal rons are born first, followed by small-diameter TrkA+Department of Anatomyand Developmental Biology, horn during the first postnatal weeks and on recent neurons2,3. Recently, an even later wave of nociceptiveWellcome Pain Consortium; data that show how that circuitry might be altered by neurons has been shown to arise from boundary capUniversity College London, sensory inputs in early life. It begins with the specifi- cells, which are neural crest derivatives that migrateGower Street, London, cation of nociceptive neurons — recent studies have down the root from the dorsal root entry zone4. AllWC1E 6BT, UK.e-mail: identified key molecular pathways that control the these neurons require neurogenin 1 or 2 (NGN1/2) genesis of spinal nociceptive circuits. It then traces the — two neuronal determination genes that encodedoi:10.1038/nrn1701 formation of functional synapses, neural circuits and basic helix–loop–helix (bHLH) transcription factorsNATURE REVIEWS | NEUROSCIENCE VOLUME 6 | JULY 2005 | 507 © 2005 Nature Publishing Group
  2. 2. REVIEWS a Nociceptor specification b Neuronal survival c Axon growth — NGN2 primarily for the generation of TrkC+ and TrkB+ neurons, and NGN1 for the generation of TrkA+ neurons5. Competitive interactions between these pre- cursors might control the final proportions of different neuronal subtypes normally produced. As the majority of CFIBRE nociceptive neurons are TrkA+ at this stage, and the non-nociceptive cutane- ous muscle afferents are TrkC+ and TrkB+ (FIG.1), the two functional subpopulations of primary sensory neurons seem to be under separate transcriptional control. There is also evidence to indicate that in thed Downregulation of TrkA e Physiological phenotype f Neuropeptide expression TrkA+ cell population future peptidergic neurons are generated before nonpeptidergic, isolectin B4- positive (IB4+) cells3, although the crucial transcrip- tion factors are unknown. The final numbers of DRG cells are determined by the balance between cell birth and subsequent cell death, and the survival of DRG cell populations is regulated by access to neuro- trophic factors6 (FIG. 1). The number of DRG neurons increases steadily until birth, and is then followed by a 15% loss during the first five postnatal days, which coincides with innervation of the skin7. Axon growth to peripheral and central targets In the rat, outgrowth of sensory axons from the DRGg Innervation density h Sensitization to peripheral and central target skin occurs before birth and reflects the two waves of neurogenesis. Inflammation In the hindlimb skin, larger-diameter A FIBRES form a cutaneous nerve plexus, and the formation of C fibres follows soon after8. Early skin nerve fibres first reach up to the epidermal surface, subsequently withdrawing as the epidermis thickens at about the time of birth, and morphologically distinct end organs develop 8,9 . Neurotrophins acting on Trk receptors have an important role in regulating Mechanoreceptor SP+ nociceptor TrkA this process (FIG. 1). Meanwhile, centrally directed Precursor dorsal root fibres reach the lumbar cord and, after Nociceptor IB4+ nociceptor Dying cell a significant delay, penetrate the grey matter. The A fibres penetrate first, at EMBRYONIC DAY (E)15–17,Figure 1 | Neurotrophins and nociceptor development. Almost every aspect of and are followed by C fibres several days later atnociceptor development is influenced by neurotrophins. a | Determination of sensory E18–20 REFS 8,10. From the outset, each DRG inner-neuron fate. Neurotrophins have an instructive role in nociceptor specification182. vates characteristic skin dermatomes and projects inb | Nociceptor survival depends on access to nerve growth factor (NGF), which is a precise somatotopic pattern in the dorsal horn11,12.produced in the target tissue and acts on the tyrosine kinase A receptors (TrkA)6,183. The Functionally, this means that the lack of RECEPTIVEunderlying mechanisms seem to require the pro-apototic B-cell leukaemia/lymphoma 2 FIELD ‘tuning’ (discussed below) — whereby the(BCL2) homologue, BAX, heat shock protein 27 (HSP27) and bone morphogenic protein(BMP)184–186. c | Neurotrophins regulate axon growth, independently of cell survival189,190. immature CNS is less able to localize stimuli to ad | A subpopulation of nociceptors downregulates the expression of TrkA receptors around particular area of the body surface — is not the resultthe time of birth, loses its NGF dependency and becomes responsive to glial cell line- of inaccurate afferent central connections. Graftingderived growth factor (GDNF), a member of the transforming growth factor-β (TGFβ) family. experiments have shown that DRGs are not matchedThese neurons form the IB4-positive (IB4+) group of nociceptors187,188. e | The physiological to particular skin targets; although the peripheral tar-properties of nociceptors are determined by neurotrophin levels during the postnatal get skin influences the pattern of projections in theperiod. NGF and neurotrophin 3 (NT3) regulate the differentiation of Aδ-fibre high-thresholdmechanoreceptors and C-fibre nociceptors. The ability of unmyelinated nociceptors to CNS, it does not direct cutaneous axons to specificrespond to noxious heat depends on exposure to NGF during the early postnatal populations of neurons in the dorsal horn13,14. Severalperiod191–193. f | Regulation of the chemical phenotype of nociceptors by neurotrophins. extracellular molecular cues might be important inThe expression of neuropeptides, such as substance P (SP) and calcitonin gene-related directing axon growth of sensory neurons, includingpeptide in TrkA-expressing neurons is triggered by peripheral target innervation194,195. the inhibitory growth cone collapsing molecule sem-g | The innervation density of nociceptors in the skin is regulated by access to the local aphorin 3A and the homeodomain protein DRG11neurotrophins NGF and brain-derived neurotrophic factor (BDNF)194–197. Neonatal skin REFS 1517. In addition, fetal DRG cells fire actionwounding during a critical neonatal period upregulates neurotrophin levels, which results inprolonged hyperinnervation of the skin24,125. h | Excess neurotrophins sensitize peripheral potentials spontaneously during this period, whichnociceptors, thereby increasing their responses to noxious stimulation, but only after might play a part in the formation of appropriatepostnatal day 10 REF. 110. synaptic connections18.508 | JULY 2005 | VOLUME 6 © 2005 Nature Publishing Group
  3. 3. FO C U S O N PA I N Functional development of nociceptors terminals cannot be detected here until P5, despite Many of the defining features of nociceptive C fibres their presence in the DRG from E18 REF. 20, which can be detected at an early embryonic age, such as the indicates that these fibres might form central con- expression of the neurotrophin receptor TrkA and nections later than peptidergic fibres29. The relatively selective binding of IB4. The capsaicin receptor TRPV1, late formation of central C-fibre synapses means that, which has a key role in detecting painful thermal and despite the ability of polymodal nociceptors to signal chemical stimuli, is expressed in a similar percentage noxious events in the periphery, central nociceptive of DRG neurons at postnatal day (P)2 as in the adult. A processing is immature in the postnatal period. This subset of these neurons might undergo the switch from is discussed in more detail below. nerve growth factor (NGF)- to glial cell line-derived Perhaps as a result of their earlier arrival in the neurotrophic factor (GDNF)-dependence that occurs dorsal horn grey matter, A-fibre terminals initially postnatally in DRG cells19 (FIG. 1). At this age, many grow beyond their final adult destination. In the adult, DRG cells also express the ATP-gated P2X3 receptor, lamina II is exclusively occupied by C fibres, but in the which is important for thermal and mechanical hyper- neonatal period it is also occupied by transient A-fibre algesia following inflammation or nerve injury. The terminals. Some, but not all, in-growing cutaneous tetrodotoxin (TTX)-resistant sodium channel Nav1.8 afferent A fibres extend dorsally right up into laminae (SNS/PN3), one of a group of voltage-gated sodium I and II to reach the surface of the grey matter, and channels that regulate neuronal hyperexcitability and only gradually withdraw over the first 3 postnatal might trigger chronic pain in adults, is expressed in weeks to terminate in laminae III and IV REFS 3032. developing C-fibre neurons by E17, and adult levels The functional class of afferent that gives rise to these can be observed by P7 REF. 20. exuberant terminals is not clear, but they are unlikely Consistent with these findings, physiological to be hair follicle afferents and might arise from theC FIBRESSmall diameter unmyelinated recordings of neonatal polymodal nociceptors reveal less well-defined A-fibre ‘pressure’ receptors that areprimary afferent sensory fibres, responses to noxious chemical, mechanical and thermal seen in newborns32–34. Their presence can be observedwith small cell bodies in the stimuli with thresholds and firing frequencies that are at the electron microscopic level35,36, and they might bedorsal root ganglion. Most are characteristic of mature C fibres, which indicates that responsible for the high incidence of Aβ-fibre-evokednociceptors. They divide into a the underlying transduction mechanisms are functional MONOSYNAPTIC responses that are seen in neurons in theneuropeptide-containing, Trkreceptor-expressing group and a from the first days of postnatal life21–23. In addition, a substantia gelatinosa in young rats37,38, and the activa-lectin IB4-binding group, group of less well-defined A-fibre ‘pressure’ recep- tion of c-fos expression by Aβ fibres in the substantiaalthough the functional tors are observed in the newborn but their incidence gelatinosa at P3 but not P21 REF. 39. On the otherimplications of this are still declines with age. In contrast to C fibres, low threshold hand, C fibres grow specifically to laminae I and IIunclear. A fibres require a longer postnatal period to acquire REF. 11, so for a considerable postnatal period theseA FIBRES their full stimulus-response sensitivity, which coincides laminae are occupied by both A- and C-fibre terminals.Large diameter myelinated with myelination and the innervation of specific end The functional effect of this changing postnatal profileprimary afferent sensory fibres, organs, such as Merkel cells. All types of afferent projec- of A- and C-fibre inputs on nociceptive processing iswith large cell bodies in the tion have lower conduction velocities at birth, but their discussed below.dorsal root ganglion. Thelargest diameter Aβ fibres are absolute receptive field sizes do not appear to changemainly low-threshold with age, despite the growth of body surface area21. Specification of dorsal horn cellsmechanoceptors, and the FIGURE 1 emphasizes the important role of neuro- The spinal cord develops along a ventrodorsal grad-smaller Aδ fibres are both trophins and Trk receptors in the growth, establishment ient such that deep dorsal horn neurons are born aftermechanoreceptors andnociceptors. of phenotype and physiological sensitivity of nocicep- motor neurons, and substantia gelatinosa neurons in tors. The early maturation of nociceptor transduction lamina II are the last to mature2. During the earlyEMBRYONIC DAY means that nociceptive activity can be transmitted to phase of neurogenesis in the mouse, interneurons are(E). These are dated from the the CNS even before birth, although any change in generated along the dorsoventral axis and settle intime of fertilization. Rat the normal neurotrophin profile during the perinatal the deep laminae of the dorsal horn. This is followedgestation is 21.5 days, mouse alittle shorter. Rats are born period could markedly alter the nature of this activity. by the development of two late-born populations inrelatively early in terms of CNS It is important to note that large increases in neuro- the superficial laminae, which can be distinguisheddevelopment and the early trophin levels follow neonatal tissue injury24. by their complementary expression of the homeoboxpostnatal period is often genes Lmx1b and Pax2 REF. 40. The expression ofparalleled with the finalgestation of development in Development of central connections homeobox gene Lbx1 is required for both the correcthumans. C-fibre projections are the last group of primary specification of substantia gelatinosa neurons and the afferents to enter the dorsal grey matter, well after appropriate sensory afferent innervation of the dorsalRECEPTIVE FIELD proprioceptive and low-threshold A fibres. In the rat horn41. Recent evidence shows that post-mitotic selec-The area on the body surface lumbar cord, the first dorsal root afferents enter the tor genes Tlx1 and Tlx3 determine excitatory gluta-that, when stimulated, evokesaction potentials in a given dorsal horn at E13, but TrkA+ C-fibre terminals are not matergic versus inhibitory GABA (γ-aminobutyricneuron. observed in lamina II until E18–19 REFS 8,10,11,25,26. acid)-mediated cell fates in the embryonic dorsal From the outset, these fibres terminate in a somato- spinal cord42. Interestingly, generation of lamina I pro-MONOSYNAPTIC topically precise manner in laminae I–II of the dorsal jection neurons is complete before that of local circuitA direct synaptic input frompre- to postsynaptic neurons horn and include those that express substance P, interneurons43, 44, which implies that direct transmis-with no involvement of calcitonin gene-related peptide (CGRP), galanin and sion of nociceptive activity from the spinal cord tointerneurons in between. somatostatin27,28. The IB4+ subset of C-fibre synaptic higher centres could develop before the onset of localNATURE REVIEWS | NEUROSCIENCE VOLUME 6 | JULY 2005 | 509 © 2005 Nature Publishing Group
  4. 4. REVIEWS a b c P1–3 P4–7 P20–25 P60–80 100 Tail 4 90 EDL Error rate of total NWR (%) 80 Reflex excitability 3 70 Reflex movement 60 Erroneus Correct 50 2 40 PER 30 1 20 10 0 0 3 10 21 0 4 8 12 16 20 24 28 Postnatal day Postnatal day d e 100 Early postnatal Mechanical threshold (g) Mechanical threshold (g) 5 80 Late 60 postnatal 2.5 40 Bilateral Unilateral 20 None 0 0 3 10 21 9 9 9 9 2 9 5 9 3. 5. 7. 9. 1. 1. –9 1. –3 –3 –3 –3 –4 –5 –3 52 Postnatal day 32 34 36 38 40 42 30 Postconceptional age (weeks) Figure 2 | Reflex modules and nociceptive responses in neonates. a | The flexion reflex response to mechanical skin stimulation decreases in amplitude, duration and sensitivity with age in rat pups. b | Cutaneous receptive fields of individual hindlimb muscles undergo postnatal tuning in rat pups56. Darker colour indicates higher sensitivity to cutaneous stimulation. EDL, extensor digitorum longus; P, postnatal day; PER, peroneus. c | Rat pup tail reflex responses to a focused laser heat stimulus become increasingly accurate with postnatal age. The graph shows the fall in error rate for three individual animals55. NWR, nociceptor withdrawal refex. d | During the first postnatal weeks in rat dorsal horn cells, cutaneous receptive fields (blue circles in inset) decrease in size (as measured by responses of the dorsal horn cells), and mechanical thresholds increase (graph)58. e | Unilateral abdominal skin stimulation in human infants evokes limb flexion, which becomes increasingly tuned with postconceptional age50. Although the oldest babies show no knee flexion (only abdominal muscle contraction) to abdominal skin stimulation, the majority of very young babies flex both their knees. At intermediate ages, unilateral knee flexion on the side of the stimulus predominates. Panel b reproduced, with permission, from REF. 55 © (2003) Society for Neuroscience; panel c reproduced, with permission, from REF. 95 © (2003) Macmillan Magazines Ltd; panel e reproduced, with permission, from REF. 50 © (2002) International Association for the Study of Pain. modulation by interneurons. At P18–27, the miniature can be evoked from E15–17 — when the first connec- excitatory postsynaptic current (mEPSC) frequencies tions are formed between A-fibre dorsal root afferents, of lamina I projection cells are five times higher than interneurons and motor neurons — and nociceptive those in interneurons45. The relatively late maturation stimulation evokes a response soon afterwards46. of interneurons means that their axodendritic growth At birth, cutaneous reflex responses are diffuse and takes place postnatally, which might be important untuned. Dorsal root stimulation evokes prominent for activity-dependent shaping of nociceptive circuits INTERSEGMENTAL REFLEXES in isolated neonatal cord, which (see below). decline in magnitude with age47. A noxious prick on the foot of a rat pup or human infant can cause move- Functional maturation of nociceptive circuits ment of the whole body and simultaneous responses The formation of synaptic connections between sen- from all four limbs, which only gradually become sory afferents and spinal cord neurons leads to the more individuated and restricted to the movement ofINTERSEGMENTAL REFLEXES first functional reflex responses to tactile and noxious an isolated leg or foot. Cutaneous reflexes have lowerMotor reponses evoked bysensory stimulation in different stimuli. Although these spinal reflexes should not be thresholds and more synchronized and prolongedspinal segments. interpreted as evidence of pain awareness or ‘feelings’, muscle contractions, which become less pronounced they are an important indication of the maturation of after 29–35 POSTCONCEPTIONAL WEEKS in the human andPOSTCONCEPTIONAL WEEKS functional neural circuits that can protect an animal P8 in the rat; they are also easily sensitized by repeatedPostconceptional age is the ageof a premature human infant from tissue damage and can trigger a range of physi- stimulation48–52. Thresholds for withdrawal from heatdated from the estimated time ological responses throughout the CNS. In isolated rat stimuli are also lower in younger animals and theof conception. lumbar cord, hindlimb responses to touching the skin sensitivity of neonatal rats in response to formalin is510 | JULY 2005 | VOLUME 6 © 2005 Nature Publishing Group
  5. 5. FO C U S O N PA I N tenfold higher than that of weanlings53,54. Until P10, the release is asynchronous and synaptic activation can- response to formalin (applied to the foot) also consists not readily evoke spike activity in vivo (FIG. 3). This predominantly of ‘nonspecific’ whole body movement, indicates that rapid nociceptive responses are largely whereas the targeted flexion, shaking and licking of the mediated through Aδ fibres in the newborn and that, paw predominates from then on. Furthermore, there is to be effective, C-fibre inputs require more sustained a high error rate in the direction of a tail flick response or widespread convergent input. to precise laser stimulation of the tail for the first 10 postnatal days, which gradually improves over three The balance of excitation and inhibition weeks55. This is consistent with the finding that the In many respects, dorsal horn cells in newborn animals receptive fields of hindlimb flexor muscles are large are more excitable than their mature counterparts. At and disorganized in young animals, such that nox- birth, the cutaneous receptive fields of dorsal horn ious stimulus-evoked limb withdrawal is not always cells, particularly those in the deep dorsal horn, are appropriate to the stimulus56 (FIG. 2). relatively larger than those in adults and rapidly These changing behaviours arise from fine-tuning decrease in size during the first two postnatal weeks58,59. of both excitatory and inhibitory synaptic connections Noxious stimulation of the skin at early postnatal ages and neuronal circuitry over the postnatal period, and (P0–P3) often results in a prolonged after-discharge the focus for such tuning has been on the interneu- — which consists of action potential activity that lasts rons of lamina II. Lamina II displays low-frequency, 30–90 s beyond the end of the stimulus — an effect that spontaneous EXCITATORY POSTSYNAPTIC CURRENTS (EPSCs) decreases in amplitude and duration with age21. The and INHIBITORY POSTSYNAPTIC CURRENTS (IPSCs) from responsiveness of newborn dorsal horn cells is likely birth, which increase markedly in frequency with to contribute to the diffuse and exaggerated reflex age. Unlike in adults, neonatal spontaneous EPSCs behaviour and disorganized reflex muscle receptive (sEPSCs) are TTX-sensitive, which indicates that fields described above (FIG. 2) and reflects the imma- spike activity is important for spontaneous glutamate turity of excitatory and inhibitory synaptic transmis- release at this time57. The earlier maturation and sion in neonatal spinal cord. The elimination of some widespread presence of functional A-fibre terminals synapses (such as the Aβ-fibre input to lamina II) and in the first postnatal weeks has a significant influence the strengthening of others (such as C-fibre inputs) on the physiological responses of dorsal horn neurons. alters the balance of postnatal transmission and might In slice preparations, a high incidence of Aβ-evoked be linked to the number of silent synapses — which monosynaptic responses relative to C-fibre inputs has express only NMDA (N-methyl-d-aspartate) receptors been observed in the substantia gelatinosa neurons of (NMDARs) — in neonatal superficial dorsal horn neu- young dorsal horn37,38. In vivo, low-intensity electri- rons66–68. Many other aspects of glutamatergic excita- cal skin stimulation — which is sufficient to recruit tory synaptic transmission, such as NMDAR channel A-fibres — or non-noxious tactile stimulation evokes opening times and the number of calcium-permeable spike activity in both superficial and deep laminae at AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole latencies that progressively decrease in magnitude and propionic acid) and kainate (KA) receptor channels variability with age. As a result, dorsal horn cell cuta- in the newborn — properties that are thought to be neous mechanical thresholds are lower and a larger crucial for growth and synaptogenesis — are develop- proportion of neurons only respond to innocuous mentally regulated in the dorsal horn and impact on stimulation and have no nociceptive input58,59. These nociceptive transmission BOX 1. properties are thought to contribute to the high level of The development of organized inhibitory connec- cutaneous reflex excitability that is described above. tions is as important as the excitatory input in nocicep- In contrast to A-fibre-evoked activity, C-fibre- tion. Recently, the need for a precise balance between evoked activity in dorsal horn cells develops gradually excitation and inhibition in the nervous system has over the postnatal period. A lower percentage of dor- been emphasized, particularly at the level of den- sal horn neurons with nociceptive inputs are observed dritic inputs, at which inhibitory synapses have been in the first week of life compared with in adults60, shown to determine the impact of adjacent excitatory and electrical stimulation of C-fibres fails to evoke a synapses only if they are co-localized on the same synchronized burst of spikes in dorsal horn neurons dendritic branch and are activated simultaneously69.EXCITATORY AND INHIBITORYPOSTSYNAPTIC CURRENTS or motor neurons58,60–63. In addition, the C-fibre irri- Like excitatory synapses, inhibitory GABA-mediated(EPSCs and IPSCs). When a tant mustard oil induces only a weak flexion reflex and glycinergic synapses are postnatally regulatedneuron is voltage clamped, ion and c-fos expression in dorsal horn neurons, despite in the dorsal horn BOX 2. Unlike in the hippocam-flow across a membrane can be its ability to excite C fibres in the skin of newborn pus, these neurotransmitters are now known to bemeasured as electric current rats64,65. However, in spinal cord slices, application of essentially inhibitory in lamina II in the newborn70while the membrane potential iscontrolled with a feedback capsaicin increases glutamate release in the superficial — topical application of GABA A (GABA type A)amplifier. Whole-cell patch dorsal horn from P0, although a significant increase antagonists in vivo causes robust disinhibition of super-clamping has extended the in the effect occurs between P5 and P10, as might be ficial and deep dorsal horn activity, as it does in adultstechnique to allow recording of expected from the proliferation of C-fibre synaptic (L. R. Bremner and M. F., unpublished observations).excitatory and inhibitorypostsynaptic currents following inputs during this period57. Therefore, at least some Indeed, the net GABA-mediated drive might even besynaptic activation of cells in a nociceptive primary afferents clearly begin to form higher in the newborn than in the adult, as the decaytissue slice or even in vivo. functional synapses before birth, but neurotransmitter time of miniature IPSCs (mIPSCs) is longer than thatNATURE REVIEWS | NEUROSCIENCE VOLUME 6 | JULY 2005 | 511 © 2005 Nature Publishing Group
  6. 6. REVIEWS Newborn Adult C-fibre inputs Inputs from C-fibre nociceptor inputs Inputs from (Weak excitation and brainstem centres (Strong excitation and abundant) brainstem centres relatively sparse) (Weak and immature) (Strong and Mature GABA and possibly better glycine signalling and targetted) Immature GABA and increased afferent glycine signalling control over inhibition? Inhibitory Inhibitory interneuron interneuron Lamina II Lamina II neuron neuron High levels of Ca2+ entry through AMPA, Mature glutamatergic kainate and NMDA and peptidergic receptors A-fibre mechanoreceptor A-fibre inputs signalling inputs (Strong excitation and (Moderate excitation but relatively abundant) relatively sparse) Excitatory Excitatory interneuron interneuron Figure 3 | Schematic diagram of the synaptic changes that take place in the superficial laminae of the dorsal horn over the first 2–3 postnatal weeks. The balance of excitation and inhibition differs in the neonatal dorsal horn compared with that in the adult. Although inhibitory transmission is present, it may be less targeted in the neonate than in the adult. This is depicted as a possible absence of specific afferent and descending control of inhibitory interneurons. In the neonate, C-fibre synaptic transmission is weak and the frequency of miniature excitatory postsynaptic currents (mEPSCs) is low. In addition, the Ca2+ entry into neurons on activation is enhanced by the unique properties and distribution of the AMPA (α-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid), kainate and NMDA (N-methyl-D-aspartate) channels in neonates, which might be important for strengthening of these synapses (see BOX 2). Transmission at inhibitory synapses in the neonate is predominantly GABA (γ-aminobutyric acid)-mediated, with little glycinergic activity. The frequency of miniature inhibitory postsynaptic currents (mIPSCs) is low, but IPSC decay times are prolonged. Several properties of GABAA (GABA type A) channels change over this period BOX 2 — for example, the intracellular concentration of Cl– is high in the newborn and declines with age, which means that, at birth, GABA can be depolarizing but still inhibitory, as the reversal potential for Cl– (ECl) is below the spiking threshold. In the neonate, descending fibres are present but inhibitory influences are weak or absent. This is probably due to low levels of the transmitter serotonin, as its receptors are functional in the early neonatal period. Immature inhibitory interneuronal connections might also be responsible. Myelinated A fibres have a strong input into neonatal superficial laminae (depicted as both mono- and polysynaptic), which declines substantially, presumably through synaptic weakening, during the postnatal period. Conversely, C-fibre synapses strengthen with age. These changes result in a shift in the balance of C-fibre to A-fibre input. The developmental changes in glutamatergic transmission BOX 2 are as important for weakening A-fibre connections as for strengthening C-fibre inputs and, possibly, for excitatory inputs to inhibitory interneurons. in adults. This is due to the production of 5α-reduced GABA-mediated activity72. Such a mechanism would neurosteroids, which confer slow kinetics on mIPSCs explain the lack of tuning in nociceptive reflex in lamina II of the neonatal spinal cord71. However, responses, the larger receptive fields and the lower it is important to note that the inhibition is almost thresholds of neonatal dorsal horn cells that are entirely GABA-mediated at this time; faster glyciner- described above (FIG. 3). With regard to this point, it gic inhibition develops later in the postnatal period70. is also interesting to note that presynaptic inhibition, This affects the temporal integration of inhibitory and as measured by dorsal root potentials, is asynchronous excitatory inputs into the neonatal dorsal horn. in the spinal cord of newborns73. Although inhibitory synaptic activity is evident in the newborn dorsal horn, this inhibition might Descending control of nociceptive circuits not be appropriately targeted and, therefore, might Descending activity from the brainstem is another not modulate excitatory inputs with precision. An factor that contributes to excitation and inhibition in example of this has been reported in visual tectal spinal nociceptive circuits. As a result of the prolonged neurons, in which the gradual reduction in receptive postnatal maturation of descending fibre systems, field size is accompanied by a transition from dispa- spinal transection before P15 has markedly less impact rate to matched topography of excitatory and inhibi- on spinal sensory circuits than it does at older ages74. tory inputs, which is itself dependent on appropriate Serotonin-containing fibres from the brain stem512 | JULY 2005 | VOLUME 6 © 2005 Nature Publishing Group
  7. 7. FO C U S O N PA I N Box 1 | Development of excitatory synaptic transmission in the neonatal dorsal horn AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) are highly expressed in the newborn dorsal horn and steadily decrease to ~20% of neonatal levels in the adult. This decrease is accompanied by changes in subunit expression, such that the glutamate RECEPTOR SUBUNITS GluR1, 2 and 4 are more highly expressed than in the adult, but that the ratio of GluR2 to GluR 1,3 and 4 is lower155. As the presence of GluR2 reduces the Ca2+-permeability of AMPARs156, this indicates greater AMPA-dependent Ca2+ influx in neonatal spinal neurons, perhaps to drive growth and synaptic plasticity. Ca2+-permeable AMPARs are expressed in GABA (γ-aminobutyric acid)-mediated and neurokinin 1-expressing (NK1+) embryonic dorsal horn neurons in culture and in the superficial laminae of neonatal spinal cord157,158. Kainate (KA) receptors also mediate excitatory glutamatergic C-fibre synaptic transmission in the neonatal spinal cord159. Subunits GluR5 and, to a lesser extent, GluR6 and 7 are expressed throughout the cord at birth, become restricted to the superficial dorsal horn by postnatal day (P)10, and are gone by P21. KA1 and KA2 are also expressed in the dorsal horn at birth but not at P21 REF. 160. Presynaptic kainate receptors are also found on C-fibre terminals of the developing rat, and the application of kainate to immature spinal cord slices decreases AMPAR- and NMDA (N-methyl-d-aspartate) receptor (NMDAR)-mediated currents161,162. Ca2+-permeable KA receptors are also expressed on isolectin B4-positive (IB4+) C fibres, but switch to a Ca2+-impermeable form early in the first postnatal week163. NMDAR concentration is high in the neonatal dorsal horn compared with that of older animals164 and the distribution is widespread over all laminae, only gradually becoming restricted to lamina II as seen in the adult. Subunit expression also changes, and although the NMDA subunit NR2B mRNA is found throughout the cord prenatally, its expression moves dorsally so that by P21 it is restricted to laminae I and II REF. 165. In many parts of the CNS, an experience-dependent postnatal shift from NR2B to NR2A expression results in an accelerated decay rate of NMDA excitatory postsynaptic currents (EPSCs), whereas EPSCs in substantia gelatinosa neurons show rapid decay time constants throughout the postnatal period166. NR2D is the most highly expressed NMDA subunit in the rat embryonic spinal cord and has slower offset times than any other recombinant receptor167,168. NMDARs in neonatal rat substantia gelatinosa neurons also have an unusually high Mg2+ sensitivity, which indicates that they have a novel stiochiometry169. Both receptor affinity for NMDA and NMDA-evoked Ca2+ influx decline with postnatal age in rat substantia gelatinosa neurons in an activity-dependent manner87. Metabotropic glutamate receptors in the spinal cord are also differentially regulated during development — the mGluR3 and mGluR5 subtypes are highly expressed at birth and this subsequently decreases during the postnatal period170,171. invade the grey matter at about the time of birth75, is also a delay in receptor maturation. Although the but the pattern and density of adult termination low levels of serotonin might limit synaptic transmis- are not achieved in the lumbar spinal cord until P21 sion, they could still influence synaptic maturation, as REF. 76. Although descending serotonin-mediated exposure to serotonin causes rapid insertion of AMPA inputs to the mature spinal cord can produce both receptors (AMPARs) into the synaptic membrane of excitatory and inhibitory effects on nociceptive neonatal substantia gelatinosa neurons68. processing, the role of this system in modulating The noradrenergic component of descending inhi-PERIAQUEDUCTAL GREYAn area of the brainstem that spinal sensory processing in the neonate is less clear. bition seems to mature before the serotonin-containingsurrounds the aqueduct Interestingly, electrical activation of the PERIAQUEDUCTAL counterpart. The expression of spinal α2-adrenergicconnecting the third and fourth GREY (PAG) region does not produce analgesia until receptor (α2-AR) peaks during the second postnatalventricles. This area projects to P21 REF. 77, and stimulation of the dorsolateral funic- week83, and α2-AR agonists selectively depress C-fibre-the medullary raphe region, ulus cannot inhibit the firing of dorsal horn neurons evoked ventral root potentials in the immature spinalwhich, in turn, sendsprojections down the until at least P10 REFS 78,79, which strongly indicates cord in vitro84 and elicit analgesia in response to noxiousdorsolateral funiculus of the that descending inhibition has little functional impact mechanical and thermal stimuli in neonatal rats85. Thespinal cord to the dorsal horn. in the neonatal dorsal horn. results of recent studies show that epidural dexmedeto-This pathway is known to In support of this, the classic biphasic behavioural midine, a potent and selective α2-AR agonist, reversesstrongly modulate spinal painprocessing. response to formalin, which, in adults, is thought to inflammatory hyperalgesia at all postnatal ages at doses arise from descending inhibition, is also not appar- that have no effect on baseline sensory processing, andRECEPTOR SUBUNITS ent in the rat pup until P15 REF. 80. Application that are lower than those required in the adult86.Ion channels are generally made of D-amphetamine at doses sufficient to enhanceup of several glycoprotein monoamine release does not affect pain behaviour Shaping of nociceptive circuitrysubunits that surround a centralpore. These subunits can confer before P10 REF. 81, which indicates that the delayed The increasing postnatal maturation of C-fibre-special characteristics on a postnatal functional maturation of this system might be evoked activity in the dorsal horn plays an impor-channel, such as increased due to low levels of serotonin (5-hydroxytryptamine, tant part in the organization of nociceptive circuitry.calcium permeability or longer 5-HT) in synaptic terminals during the early post- Neonatal destruction of C fibres by systemic admin-opening times. The subunits ofmany channels change with natal period. However, intrathecal administration of istration of the neurotoxin capsaicin prevents ordevelopment, thereby altering serotonin agonist does not depress the formalin test delays the development of several synaptic processesthe channel properties. response until P10 REF. 82, which indicates that there in lamina II. These include a delay in the postnatalNATURE REVIEWS | NEUROSCIENCE VOLUME 6 | JULY 2005 | 513 © 2005 Nature Publishing Group
  8. 8. REVIEWS Box 2 | Development of inhibitory transmission in the neonatal dorsal horn GABA (γ-aminobutyric acid) synthesizing enzymes, the glutamic acid decarboxylases 65 and 67 (GAD65/67), are expressed in the spinal cord from an early embryonic stage172, but GABA is not expressed in the substantia gelatinosa until embryonic day (E)17–E18 REF. 173. GABA-positive spinal neurons increase in the first postnatal weeks and then decrease towards adulthood174. A mature pattern of glycine expression is present before birth, mainly in the deep dorsal horn, which coincides with the presence of glycine transporter GLYT2 REF. 175. Nevertheless, newborn (postnatal day (P)0–P1) lamina II neurons show only GABA miniature inhibitory postsynaptic currents (mIPSCs) despite possessing functional glycine receptors (GlyRs)70. After P8, mixed GABAAR (GABA type A receptor)–GlyR mIPSCs are observed. These are subsequently downregulated so that mIPSCs at older (P>30) ages are mediated by either GlyRs or GABAA receptors (GABAARs), but not both14. Neonatal GABA mIPSCs have long decay times and the rate of GABA mIPSC decay in laminae I–II neurons increases fourfold between P8 and P23, which was recently attributed to a tonic production of 5α-reduced neurosteroids in the immature dorsal horn71. GABAAR activation in the developing CNS can be depolarising rather than hyperpolarising, due to high intracellular Cl– concentrations ([Cl]i) in immature neurons, which give rise to a Cl– REVERSAL POTENTIAL (ECl) that is more positive than the resting potential and, in some cases, the action potential threshold. The onset of expression of the K+–Cl– cotransporter KCC2 leads to a decrease in [Cl]i during the postnatal period, which leads to a negative shift in ECl, such that GABA and glycine become progressively hyperpolarising and inhibitory36. In cultured embryonic dorsal horn cells, GABAAR or GlyR activation results in membrane depolarization and elevated levels of intracellular Ca2+ REFS 176,177. A subset of newborn superficial dorsal horn neurons in spinal cord slices depolarize in response to GABAAR activation, a response that becomes exclusively hyperpolarizing by P6–P7. However, ECl is consistently more negative than the action potential threshold, which indicates that depolarizing GABA responses will still inhibit the firing of newborn superficial dorsal horn neurons70. GABAAR depolarization mediates Ca2+ influx through N-type and L-type voltage-gated Ca2+ channels176 and relieves the Mg2+ block of NMDARs178, which might be important for synapse formation and remodelling168. All three GABABR (GABA type B receptor) subunits (GABABR1a, 1b, and 2) and the GABACR (GABA type C receptor) subunits, rho1 and rho2, are also expressed in the immature spinal cord179,180. Significant GABABR- mediated presynaptic inhibition of primary afferent-evoked excitatory postsynaptic currents (EPSCs) is observed from birth, but the effects of postsynaptic GABABR increase over the first postnatal week70,181. reduction of the NMDA-evoked calcium influx and abdominal withdrawal reflexes of preterm human in lamina II cells 87 , a failure of A-fibre synaptic infants show an analogous lack of tuning, and it would withdrawal from lamina II cells88,89, a failure in the be interesting to know if these are also influenced by organization of expression patterns of neurokinin 1 tactile stimulation49,50. (NK1) receptors90, abnormal maturation of GABA- The mechanism that underlies this A-fibre- mediated and descending inhibition91,92, and disor- induced synaptic tuning is not known. Repeated ganized somatotopic maps93. afferent stimulation might convert silent synapses in However, under normal physiological circumstances, dorsal horn cells to functional ones by rapid insertion activation of nociceptors is rare in the first postnatal of AMPARs68. The postnatal synaptic elimination of weeks and, in any case, C-fibre-evoked spike activity exuberant A-fibre inputs, reduction of receptive field is weak at this time. This is an important considera- size and the increase in mechanical threshold of the tion, as spike activity is required for enhanced neuronal dorsal horn are all activity-dependent processes, asHEBBIAN WEAKENING protein synthesis and EPSCs in the absence of spiking they can be prevented by chronic, local blockade ofHebb proposed that if a neuron, can actually inhibit protein synthesis94. On the other NMDARs in the dorsal horn30. Intrathecal applicationA, took part in firing anotherneuron, B, then a plastic change hand, A-fibre inputs evoke plenty of spike activity in the of low doses of the NMDAR antagonist MK801 in P0would occur in the synapse newborn dorsal horn, and repetitive A-fibre stimulation rat pups leads to greater A-fibre-evoked responses inbetween neurons A and B, such causes sensitization of dorsal horn cells in the neonate, substantia gelatinosa neurons and reduced mechani-that the connection between A which takes the form of a build-up of background activ- cal thresholds compared with adults, whereas theand B would be strengthened.This has been extended to ity in the cells and post-stimulus after-discharge that response to C-fibre inputs and noxious heat thresholdinclude the opposite effect — lasts for a matter of minutes60. Therefore, spike activity- are unaffected (FIG. 4). It is possible that the postnatalthat is, failure to take part in dependent shaping of nociceptive circuits might arise developmental increase in firing frequency of A-fibrefiring leads to synaptic from low-threshold A-fibre activity. This is supported mechanoreceptors33 might lead to a gradual mismatchweakening. by the finding that postnatal tuning of the nociceptive in timing of pre- and postsynaptic spikes in lamina IIREVERSAL POTENTIAL tail reflex is not affected by daily noxious stimulation, cells, which are increasingly driven by low-frequencyThe membrane potential at but can be prevented by blocking low-intensity tactile C-fibre inputs. This could lead to a HEBBIAN WEAKENINGwhich chemical and electrical inputs from the tail with local anaesthetic during the of synapses between A-fibre terminals and lamina IIdrive are equal and opposite, so critical ten-day period. So, low-intensity tactile inputs, cells, perhaps through long-term depression (LTD)-there is no net flow of ionsacross the membrane. The such as those that arise from spontaneous twitching type mechanisms. Low-frequency Aδ-fibre stimula-direction of flow reverses above during sleep, have been proposed to be responsible for tion does produce a robust, NMDAR-dependent LTDand below this potential. shaping nociceptive circuits in early life55,95,96. The limb in young lamina II cells97.514 | JULY 2005 | VOLUME 6 © 2005 Nature Publishing Group
  9. 9. FO C U S O N PA I N a b c Lumbar spinal Naïve Vehicle MK801 cord L5 DRG L4 DRG Vehicle Sciatic p < 0.001 nerve ELVAX implant 125 *** 100 Distance (µm) 150 75 Neuronal cell MK801 100 count 50 50 CGRP 25 IB4 0 NeuN 0 Naïve Vehicle MK801 Naïve Vehicle MK801 d p < 0.05 e p < 0.001 40 100 * *** f Receptive field area 75 Evoked spikes 30 Mechanical withdrawal 30 Thermal withdrawal p < 0.05 10 threshold (g) 20 50 7.5 latency (s) 20 * 5 10 25 10 2.5 0 0 0 0 Vehicle MK801 Vehicle MK801 Naïve Vehicle MK801 Naïve Vehicle MK801 n = 11 n = 12 n = 29 n = 28 Figure 4 | Activity-dependent development in spinal cord sensory connections. a | Local, low-dose chronic NMDA (N-methyl-D-asparate) antagonist MK801 is applied to the dorsal surface of the rat spinal cord at birth and the dorsal horn circuitry is examined six weeks later. An ELVAX implant provides slow release of MK801 or vehicle. L4/L5 DRG, fourth and fifth lumbar dorsal root ganglion. b | The number of dorsal horn cells and termination of C fibres in lamina II are unaffected. CGRP, calcitonin gene-related peptide; IB4, isolectin B4; NeuN, neuronal nuclear antigen. c | A-fibre terminals are still present in lamina II in MK108- treated animals. The graph shows the length of A-fibre terminals as measured from the surface of the dorsal horn. d | The activity that is evoked by dorsal horn A fibres is enhanced in MK108-treated animals. e | Dorsal horn receptive fields are larger in MK108-treated animals. f | The thresholds for mechanical behavioural reflex are lower in MK108-treated animals, but thermal thresholds are normal. Adapted, with permission, from REF. 30 © (2002) Blackwell Publishing. The effects of early tissue damage produced in young animals from P3 or earlier, although Short-term effects. Nociceptive activity might not its magnitude is not always as great as in adults54,101–104. be a normal physiological event for newborn mam- In vivo electrophysiological studies show sensitisation of mals, but in the event of tissue injury there is strong dorsal horn cells after carageenan inflammation of the C-fibre activation over a prolonged period of time. hindpaw at all postnatal ages, although the exact pattern C-fibre nociceptors can be sensitized by inflammatory of effects is age-dependent. Spontaneous activity and chemicals from before birth23, which further increases the magnitude of responses increase from the earliest the afferent barrage after injury. Almost as soon as postnatal age that has been studied (P3), but inflamma- peripheral C-fibre stimulation begins to evoke spike tion-induced expansion of mechanical receptive field responses in dorsal horn cells, repetitive stimulation size is not observed until at least the second postnatal evokes an NMDAR-dependent ‘wind-up’, in which week59. Hypersensitivity to tissue damage can also be the response amplitude increases with each subse- measured in human infants, in which ‘tenderness’ or a quent stimulus58,98,99. Long-term potentiation (LTP) of fall in reflex thresholds is established for days or weeks in lamina II cells has also been observed after repetitive the presence of local or deep visceral tissue injury49,50,105. high-frequency C-fibre stimulation of laminae I–II The effect is small in the youngest infants and increases cells in juvenile rats100. ‘Wind-up’ and sensitization with age in both rats and humans. Secondary hyper- are known to be central mechanisms that underlie algesia, which spreads into an area that surrounds the injury-induced pain. original injury site, seems to develop later than primary Peripheral tissue injury not only evokes immediate hyperalgesia in newborn rats50,73. nociceptive responses but can also lead to a state of The postnatal maturation of hyperalgesia might hyperalgesia and allodynia, in which noxious responses relate to the development of signalling by substance P, are enhanced and can be evoked by previously non- which is known to be important for this form of central noxious stimulation. Hyperalgesia that lasts for minutes sensitization. Substance P is released in very low quanti- (mustard oil), hours (formalin), days (carageenan) and ties before P10 REF. 64, although it is clearly involved in weeks (complete Freunds adjuvant, CFA) can all be C-fibre-evoked LTP in lamina I projection neurons inNATURE REVIEWS | NEUROSCIENCE VOLUME 6 | JULY 2005 | 515 © 2005 Nature Publishing Group