Peripheral sensitization Th e response to a given nociceptive stimulus can change with time , and sensitization of nociceptive pathways provides further warning and encourages protect ion of the injured part (Woolf and Ma, 2007). Hyperalgesia refers to an increased response to a noxious stimulus (increased response to suprathreshold stimuli) and allodynia refers t o pain or nociceptor fi ring in response to a previously non-noxious stimulus (reduction in threshold and spontaneous activity). Primary hyperalgesia or peripheral sen- sitization is the result of altered nociceptor sensitivity, with an increased response to thermal and me chanical stimuli within the area of tissue damage. By contrast, secondary hyperalgesia refe rs In \202 ammation Peripheral infl ammation results in pain, heat, and redness (due to vasod ilation), and swelling (due to plasma extravasation). An \200\230infl ammatory soup \200 \231 that is \200\230tasted \200\231 by receptors on nerve terminals (Woolf and Ma, 2007) is made up of multiple mediators includ- ing: prostaglandins (Kawabata, 2011), leukotrienes (Noguchi and Okubo, 2011), substance P, nitric oxide, cytokines, chemokines, bradykinin, s erotonin, histamine, tumor necrosis factor (TNF ) and neurotrophic factors (e.g. NGF). F ollowing release from the nerve terminal, or from infl ammatory cells or damaged tissue, CHAPTER 6 nociceptive signaling in the periphery and spinal cord 57 these mediators activate and/or sensitize the nociceptor terminal by acting on specifi c se nsory receptors that include (Woolf and Ma, 2007; Linley et al., 2010): et al., 1997; Suzuki et al., 2002). Importantly, the functional prop- erties of the SDH net work are highly age-dependent. As a result, understanding how the immature SDH is organized at a cellular and molecular level may yield new insight into novel approaches to regulate the ascending fl ow of noxious information to the brain, and resultant pain perception, in infants and children. \227\206 G protein-coupled receptors (e.g. bradykinin B1 and B2; pro- tease-activated receptors, PAR 1 \200\2233; histamine receptors). \227\206 Receptor tyrosine kinases (e.g. growth factor receptors such as trkA). Electrophysiological properties of neonatal dorsal horn neurons Extracellular single-unit r ecordings in intact, anesthetized rodents clearly demonstrated that immature dorsal horn ne urons are char- acterized by low thresholds, large peripheral receptive fi elds, and prolon ged action potential aft er-discharges compared to adult dorsal horn cells (Fitzgerald, 198 5; Torsney and Fitzgerald, 2002). Repetitive low-threshold stimulation also leads to a prog ressive sensitization of neonatal dorsal horn neurons, which disappears by P21 (Jennings an d Fitzgerald, 1998). Notably, these same functional properties also characterize nociceptiv e withdrawal refl exes in the newborn rodent (Schouenborg, 2003), kitten (Ekholm, 1967), an d human (Andrews and Fitzgerald, 1994; Andrews et al., 2002). Th e developmental alteration s in the response of dorsal horn neurons following stimulation of their peripheral receptiv e fi elds may be explained by three factors: \227\206 Ionotropic receptors/ion channels, such as TRP channels (Patapoutian et al., 2009 ), purinergic receptors (Burnstock, 2006), and acid-sensitive channels. Infl ammatory mediators initiate intracellular signaling cas- cades, involving activation o f a range of protein kinases (Bhave and Gereau, 2004; Hucho and Levine, 2007) and mediate i ncreased sen- sitivity by: \227\206 Rapid response post-translational changes which directly modu- late ion channel a ctivity, change membrane potential, or increase insertion of receptors into the membrane (B have and Gereau, 2004). \227\206 Alterations in transcriptional programs and changes in gene expression resulting in longer-term altered sensitivity (Linley et al., 2010), such as changes in the expression and transportation of pro-nociceptive molecules (e.g. substance P, NGF) and ion channels. \227\206 Developmental changes in sensory transduction and/or the func- tional properties of primary aff erent neurons. Infl ammatory hyperalgesia occurs from early development. Responses to thermal stimuli are enhanced following applica- tion of infl ammatory mediators in skin-nerve preparations from embryonic chick (Koltzenburg and Lewin, 1997). Infl ammatory stimuli, such as carrageenan and complete Freund \200\231s adjuvant (CFA), produce hyperalgesia in neonatal rat pups, an d the degree and duration of response varies with the dose and type of agent (Ren et al., 2 004; Walker et al., 2003). Hindpaw infl ammation also accelerates the postnatal upregulatio n of IB4+ve neurons in the DRG (Beland and Fitzgerald, 2001). Although the pattern of respo nse changes with age, hindpaw formalin injection also pro- duces a signifi cant behavioral response in rat pups (Guy and Abbott, 1992). \227\206 Age-dependent changes in the intrinsic membrane proper- ties of dorsal horn cells which infl uence the level of neuronal excitability. \227\206 Th e maturation of excitatory and inhibitory synaptic inputs onto dorsal horn neu rons. Th is section of the chapter will focus on the last two factors. Intrinsic excitability of developing SDH neurons An important feature of developing CNS net works is spontaneous activity (SA), which can be generated by a variety of mechanisms (Blan kenship and Feller, 2010) and contributes to both early cir- cuit formation (Tritsch et al. , 2007) and the subsequent establish- ment of appropriate sensory maps (Kandler et al., 200 9). SA in the newborn SDH is predominantly localized to lamina I, and is then downregulated with age (J. Li and Baccei, 2011a). During early life, a subset of glutamatergic interneur ons within lamina I func- tion as \200\230pacemaker \200\231 neurons that are capable of g enerating oscil- latory burst-fi ring. Th is is independent of synaptic transmission within the network and instead relies on a combination of intrin- sic voltage-gated conductances (including persistent Na + currents and high-threshold N-type and L-type Ca 2+ channels). A s these pacemaker neurons are innervated by nociceptive aff erents and project throughout t he dorsal \200\223ventral axis of the immature spinal cord, they may provide an endogenous source of activity to the developing sensorimotor circuits mediating spinal nociceptive ref l exes (J. Li and Baccei, 2011a). Alternatively, they may serve to synchronize the fi ring of ascending spinal projection neurons in the neonate. In this context, burst-fi ring also characterizes soma- tosensory processing in the developing human brain (Fabrizi et al., 201 1). Th e acute and potential long-term eff ects of neonatal injury in diff erent laboratory mod els are further discussed by Walker (Chapter 3, this volume). Nociceptive processing in the spinal cord Th e processing of noxious information by the CNS begins in the superfi cial dorsal horn (SDH) of the spinal cord (laminae I \200\223II), wh ich receives direct projections from nociceptive primary aff er- ent neurons (A and C fi bres) with cell bodies in the DRG. Th e vast majority (>95%) of neurons within the SDH cor respond to propriospinal and local circuit interneurons (Bice and Beal, 1997a, 1997b). As a result, the SDH network has the ability to integrate sensory inputs, which vary in modalit y, intensity, and location, and transform them into an output signal, which ascends to the brain. Th is output of the SDH circuit is conveyed by a small percentage of lamina I neuron s ( c .5%) which project to multiple supraspinal sites (Spike et al., 2003) and are known t o be instrumental for the gen- eration of chronic pain following tissue or nerve injury (Ma ntyh SECTION 2 biological basis of pediatric pain 58 Th e morphological and intrinsic membrane properties of SDH neurons appear to be developmen tally regulated in a cell-type dependent manner. synaptic contacts in the SDH during early life (Coggeshall et al., 1996; Fitzgerald et al., 1994). Over the fi rst 3 postnatal weeks, the majority of A fi bres gradually retract to o ccupy deeper laminae in the dorsal horn, via activity-dependent mechanisms (Beggs et al., 2 002). Some controversy exists regarding the extent to which these exuberant A-fi bre termin als refl ect low-threshold A -fi bre inputs; at least some correspond to myelinated high -threshold mech- anoreceptors (Woodbury and Koerber, 2003), and a proportion continue to pr oject into lamina I-II throughout life (Boada and Woodbury, 2008). Regardless of whether A fi bres make direct synaptic contacts onto laminae I and II neurons or communicate with these cells via polysynaptic pathways, signifi cant evidence points to enhanced A input to the SDH during the early postnatal period: \227\206 Supraspinal projection neurons undergo their axonal and den- dritic development p rior to birth (Bicknell and Beal, 1984), but ascending spinothalamic tract neurons increase in diameter dur- ing the fi rst postnatal week (Davidson et al., 2010). \227\206 Presumptive interneurons within the SDH exhibit extensive growth and reorganizati on of their dendritic trees during early life (Bicknell and Beal, 1984). \227\206 Th e general population of laminae I and II neurons (mostly cor- responding to in terneurons) demonstrates an age-dependent hyperpolarization of the resting potential, decre ase in membrane resistance and elevation in rheobase (amount of current needed to evoke an action potential), contributing to a decrease in excit- ability with maturation (J. Li and Baccei, 2011a; Walsh et al., 2009). \227\206 A fi bre stimulation evokes the expression of c-fos (a marker of neuronal acti vation) in laminae I and II at P3 but not at P21 (Jennings and Fitzgerald, 1996). \227\206 Patch clamp studies, which allow assessment of synaptic currents at a cellular le vel, have demonstrated an increased prevalence of polysynaptic A -input to neonatal GABA ergic neurons within the SDH (Daniele and MacDermott, 2009). \227\206 Lamina I neurons projecting to the parabrachial nucleus and periaqueductal gray ( PAG) diff er from surrounding interneurons as their passive membrane properties and levels of spontaneous activity are stable during the fi rst 3 weeks of life (J. Li and Baccei, 201 2). \227\206 Repetitive stimulation of A fi bres prior to P21 sensitizes neona- tal dorsal horn neurons (Jennings and Fitzgerald, 1998). While the intrinsic fi ring properties of a neuron clearly depend on the expression of both voltage-gated and voltage-independent (i.e. \200\230leak \200\231) ionic conductances, su rprisingly little is known about how age infl uences the expression of the underlying ion c hannels within developing spinal nociceptive circuits. Voltage-gated Na + currents are upre gulated postnatally in the axonal (but not somatic) membrane of laminae I and II neurons (S afronov et al., 1999). Th is may occur in the absence of a signifi cant shift in the Na + c hannel isoforms expressed within the region, as Na v 1.2 and Na v 1.3 are the dominant isof orms in rat laminae I and II neurons at both P6 to P9 and P25 to P30 (Hildebrand et al., 20 11). In addition, these neurons demonstrate an age-dependent increase in the levels of fast , A-type voltage-gated K + currents, which may contribute to the observed rise in the preva lence of SDH neurons which exhibit delayed fi ring in response to intracellular current inj ection (Walsh et al., 2009). Meanwhile, although \200\230leak \200\231 currents (which can result from any ion channel open at the resting membrane potential) strongly modulate neur onal excitability in many areas of the CNS and can be regulated by a host of factors such a s neurotransmitters, pH or anaesthet- ics (Goldstein et al., 2001), there are no data curre ntly available regarding the molecular basis for these currents in developing SDH neurons. As a result, less intense or low-threshold stimuli can elicit activation of neurons in the spinal cord in early life. Small-diameter sensory aff erents (A and C fi bres) grow into the rat dorsal horn at E19 (Fitzgerald, 1987a) and likely have estab- lished a mature pattern of projections to the s ubstantia gelatinosa (SG) by birth (Fitzgerald and Swett, 1983). However, the matura- tion of C-fi bre synaptic connections within the developing dorsal horn is delayed: \227\206 Electrical activation of high-threshold aff erents fails to produce signifi cant spike discharge in many newborn dorsal horn neu- rons (Fitzgerald, 1985, 1988). \227\206 Chemical stimulation of C-fi bres fails to evoke nociceptive with- drawal refl ex es or neurogenic oedema until the second postnatal week (Fitzgerald and Gibson, 1984). \227\206 Ultrastructural studies demonstrate increasing numbers of cen- tral terminals cor responding to fi ne-diameter primary aff erents from P5 to P20 (Pignatelli et al., 1989). \227\206 Capsaicin elevates glutamatergic signaling within the SDH to a greater extent at P10 compared to P0 (Baccei et al., 2003). Understanding how the expression of voltage-dependent and \200\230leak \200\231 channels i n the SDH varies as a function of both age and spe- cifi c cell type (i.e. GABAergic versus glutamatergic, interneuron versus projection neuron, etc.) could allow novel pharmacologic al strategies to target the output of spinal pain pathways in an age- specifi c manner. Excitatory synapses in the developing SDH All sensory neurons release glutamate from their central terminals in the spinal cord. Activation of ionotropic (AMPA, NMDA and kainate ion channel receptors) and/or metabotropic (mGluR, which signal via coupling with G-proteins), glutamate receptors results in membrane depolarization and increased excitability in dorsal horn neurons (H. J. Hu et al., 2007; P. Li et al., 1999; Yoshimura and Jessell, 1990). Th e functional properties of glutamate receptors depend on their subunit composition, and the relative expression of mGluR (Berthele et al., 1999; Valerio et al., 1997) and AMPAR subun its (Jakowec et al., 1995) within the dorsal horn vary accord- ing to postnatal age. Maturation of primary a \200 erent input to the developing SDH Large myelinated A-fi bre p rimary aff erents penetrate the rat dor- sal horn in a somatotopically appropriate manner a t embryonic day (E) 15 (Mirnics and Koerber, 1995), and make functional CHAPTER 6 nociceptive signaling in the periphery and spinal cord 59 \227\206 GluR2 reduces the Ca 2+ -permeability of AMPARs (Hollmann et al., 1991; Washburn et al., 1997). As neonatal rat SG neurons express a lower ratio of GluR2 to GluR1, 3, and 4 (Jakowec et al., 1995), AMPAR-dependent Ca 2+ infl ux may be increased in neo- natal spina l neurons, increasing excitability (Hartmann et al., 2004). either GABA A R-only or GlyR-only mIPSCs are observed. Th is shift results from an age-depe ndent reorganization of receptors in the postsynaptic membrane (Keller et al., 2001). Inhibition versus excitation In mature neurons, the activity of the potassium-chloride (K + -Cl \210\222 ) co-transporter KCC2 maintains low levels of intracellular Cl \210\222 . F ollowing activation of the GABA A R or GlyR and opening of the associated chloride channel, Cl \210\222 fl ows into the cell, the membrane is hyperpolarized, and the neuron inhibite d (Rivera et al., 1999). However, in many parts of the CNS, a low level of KCC2 during the neonatal period results in high levels of intracellular Cl \210\222 and a reversal potenti al for Cl \210\222 ions (E Cl ) that is more positive than both the resting potential and action potential (AP) threshold (Ben- Ari, 2002). As a result, following GABA A R (or GlyR) activation, Cl \210\222 moves out of the cell (down its concentration gradient), spike di scharges can be evoked, and excitation (rather than inhibition) results in the developing n etwork (Ben-Ari et al., 1989). Neonatal SDH neurons can depolarize in response to GABA A R activation (Baccei and Fitzgerald, 2004), and do not attain a mature Cl \210\222 extru- si on capacity until the third postnatal week (Cordero-Erausquin et al., 2005). Th is raises a critical question of whether GABAergic signaling within newborn spinal pain circuits is ex citatory or inhibitory in nature. \227\206 NMDARs expressed by neonatal SDH neurons have a higher Mg 2+ sensitivity and grea ter affi nity for NMDA (Hori and Kanda, 1994; Green and Gibb, 2001). Th is suggests a disti nct NMDAR composition during early life, but direct evidence is presently lacking. \227\206 Synapses expressing only NMDARs (referred to as \200\230silent \200\231 syn- aps es due to the block of the NMDAR by extracellular Mg 2+ at resting membrane potentials) are present in laminae I and II at early postnatal ages (Baba et al., 2000; Bardoni et al., 19 98; P. Li and Zhuo, 1998). \200\230Silent \200\231 synapses have been observed at high- th reshold A fi bre synapses onto NK1R-expressing lamina I neu- rons as late as P19 to P24 (Torsney, 2011). However, since 93% to 94% of glutamatergic synapses within laminae I and I I of the neonatal rat express AMPAR subunits (Yasaka et al., 2009), the overall importance of \200\230silent \200\231 synapses for nociceptive processing within the developing SDH r emains unclear. Synaptic inhibition within the developing SDH GABA and glycine GABA and glycine serve as th e major inhibitory neurotransmitters in the spinal dorsal horn via activation of two ligand -gated Cl \210\222 chan- nels (the GABA A R and glycine receptor), and are predominantly r eleased from local interneurons, although descending inhibitory pathways from the brainstem may also include GABAergic and gly- cinergic projections (Kato et al., 2006). Th e number of GABAergic neurons within the rodent SDH peaks during the fi rst two postna- tal weeks (D ougherty et al., 2009; Ma et al., 1992; Schaff ner et al., 1993) before declining to approx imately 30% of the overall popu- lation during adulthood (Todd and Sullivan, 1990). Glycine rgic neurons represent a subset of GABAergic neurons throughout embryonic and postnatal dev elopment (Berki et al., 1995; Huang et al., 2008; Todd and Sullivan, 1990). GABA and glycin e are trans- ported into the same synaptic vesicles by the vesicular inhibitory amino acid transporter (VIAAT) and co-released in the spinal cord, resulting in mixed GABA A R-GlyR mi niature inhibitory post-syn- aptic currents (IPSCs; Jonas et al., 1998). Intrathecal administration of GABA A R antagonists (gabazine and bicuculline) at P3 signifi cantly increased mechanical and thermal withdrawal thresholds, while the same antagonists at P21 decreased thresholds (Hathway et al., 2006). Th e GABA A R agonist mida- zolam sensi tized nociceptive withdrawal refl exes in the neonate, but increased threshold at older age s (Koch et al., 2008). At fi rst glance, these results appear to support the notion that GA BA acts as an excitatory neurotransmitter within the newborn SDH. However, other evidence s uggests that GABA has inhibitory actions in the SDH from birth. In vitro patch clamp studie s demonstrated that E Cl was consistently more negative than spike threshold even in neonat al SDH cells that depolarized in response to GABA, suggest- ing that GABA A R activation wo uld ultimately limit action potential discharge and inhibit these neurons (Baccei and Fitzg erald, 2004). In vivo recordings from neonatal SDH neurons show that spinal application of gabazine enlarges receptive fi elds and enhances aff erent-evoked discharge from the fi rst days of life (Bremner et al., 2006) although spinal inhibitory circuits may be poorly tune d in the neonate (Bremner and Fitzgerald, 2008). Collectively, these results suggest that G ABAergic synaptic inhibition in the SDH is functional from birth. GABAergic signaling dominates within lamina II of the neo- natal spinal cord (Baccei and Fi tzgerald, 2004). Glycinergic mIP- SCs are absent in these cells during the fi rst postnatal week, but these neurons respond robustly to exogenous glycine, suggesting that functional glycine receptors (GlyRs) are present but are not yet clustered at appropriate synaptic loc ations. Th is may refl ect a reduced expression of gephyrin, a membrane protein known to in teract with both GlyRs and the cytoskeleton in order to \200\230trap \200\231 the GlyR at synaptic sites in the membrane (Charrier et al., 2010; Kirsch et al., 1993). Th is apparent discrepancy can be explained if the excitatory actions of GABA on refl ex se nsitivity are not the direct result of its actions in the dorsal horn, but also involve mod ulation by suprasp- inal activity. Following transection of the neonatal spinal cord, intra thecal GABA A R antagonists facilitate nociceptive withdrawal refl exes in a similar manner as at P21, and gabazine also enhances primary aff erent-evoked ventral root potentials in the isolated neo- natal spinal cord (Hathway et al., 2006). Th is supports a role for supra spinal structures in modulation of neonatal cutaneous refl ex sensitivity. While refl ex be havior undoubtedly provides insight into how the nervous system processes noxious informati on, meas- uring only the motor output of the spinal cord network may not specifi cally indi cate the level of excitability within the ascending sensory pathways that project to the co rtex. From P8, mixed GABA A R-GlyR mIPSCs are apparent in lamina I-II but are subsequently modula ted in a regionally-specifi c man- ner. Within lamina I, the mixed responses are replaced b y pure GlyR-mediated mIPSCs by P23, while in mature lamina II neurons SECTION 2 biological basis of pediatric pain 60 Signal processing in the developing SDH under pathological conditions Understanding pain me chanisms in early life requires additional knowledge of how tissue and nerve damage modulat e both the intrinsic membrane properties of immature dorsal horn neurons as well as their s ynaptic inputs. Persistent e \200 ects of early tissue injury on SDH network function Tissue damage during a critical period of early life can evoke long- term changes in pain sensitivity in rodent s, with many studies reporting a baseline hypoalgesia accompanied by an exaggerated hyperal gesia following re-injury during adulthood (see Walker, Chapter 3, this volume). Th ese pro longed changes in nociceptive processing could, in part, refl ect long-term alterations in the func- tion of spinal pain circuits, as brief infl ammation of the neonatal hindpaw is k nown to result in signifi cant changes in gene expres- sion within the adult dorsal horn (R en et al., 2005). However, the extent to which early tissue damage modulates intrinsic memb rane properties and synaptic function within the mature SDH network remains unclear. Peripheral infl ammation signifi cantly increases spontane- ous fi ring and receptive fi el d size in neonatal dorsal horn neu- rons within hours aft er the injury (Ririe et al., 2008 ; Torsney and Fitzgerald, 2002), which at least in part results from an injury- evoked enha ncement in the intrinsic excitability of immature dorsal horn cells (Rivera-Arconada and Lo pez-Garcia, 2010). However, infl ammation may evoke biphasic changes in the mem- brane prop erties of developing SDH neurons, as this short-term enhancement in intrinsic excitability is followed (at c .20 hours post-injury) by an upregulation of sustained voltage-gated K + cur- rents and reduction in excitability (Rivera-Arconada and Lopez- Garcia, 2010). Meanwhi le, neonatal sciatic nerve injury failed to alter neuronal excitability in the SDH, althoug h it did unmask increased sensitivity to the pro-infl ammatory cytokine TNF (J. Li et al ., 2009b). Th e majority of behavioral studies have identifi ed the fi rst post- natal week as the \200\230critical period \200\231 during which tissue damage is capable of altering pain sen sitivity throughout life (LaPrairie and Murphy, 2007; Ren et al., 2004; Walker et al., 2009 ). Th is correlates well with the ages at which tissue damage elevates glutamatergic signal ing within the SDH (J. Li and Baccei, 2009; J. Li et al., 2009a). Nonetheless, the mechanis tic basis for the critical period within the developing SDH currently remains unknown. One possibility is that the gradual maturation of inhibitory synaptic transmission within the S DH (Baccei and Fitzgerald, 2004; Cordero-Erausquin et al., 2005; Ingram et al., 2008; Kelle r et al., 2001) is involved in terminating the critical period (Hensch, 2004). Closure of t he criti- cal period also coincides with the onset of myelination in the spinal cord (Kapfh ammer and Schwab, 1994). CNS myelin contains pro- teins that potently inhibit axonal growt h (F. Hu and Strittmatter, 2004), and enhanced collateral sprouting of nociceptive fi bres has been observed in the mature dorsal horn in the presence of reduced myelination (Schwegl er et al., 1995); and experience-dependent plasticity in the visual cortex persists through out life in mice lack- ing normal myelin-derived signaling at the Nogo receptor (McGee et a l., 2005). Future identifi cation of factors responsible for closing the critical period fo r plasticity within developing spinal pain cir- cuits may identify potential strategies to limit the long-term eff ects of neonatal tissue damage. Th e eff ects of peripheral tissue and nerve damage on synap- tic function within spinal no ciceptive networks are highly age- dependent. Tissue damage during early postnatal developm ent evokes an activity-dependent elevation in glutamate release within the SDH (J. Li and B accei, 2009; J. Li et al., 2009a). Synaptic inhibition was not signifi cantly aff ected (J. Li and Baccei, 2009), which is in contrast to the adult dorsal horn where a reduction in t he effi cacy of glycinergic inhibition is thought to make a key con- tribution to infl amma tory hyperalgesia (Harvey et al., 2004; Hosl et al., 2006; Muller et al., 2003). Similarly, while peripheral nerve injury compromises GABAergic inhibition in the adult (Moore et al., 2002; Scholz et al., 2005), it had no signifi cant eff ects on synaptic transmission withi n lamina II in the neonate (J. Li et al., 2009b). However, it remains possible that injury- evoked changes in the expression of KCC2 (and thus Cl \210\222 homeostasis) occur within t he neonatal SDH, as reported previously in the adult (Coull et al., 2003; Zhang et al., 200 8), which could reduce the functional strength of synaptic inhibition within immature spina l nociceptive circuits. Conclusions and clinical implications Nociceptive signaling in the periphery and spinal cor d in early life has the following features: Injury-induced facilitation of excitatory signaling is also asso- ciated with an increased effi cacy of nociceptive synapses within the immature SDH. Surgical incision selectively en hanced gluta- matergic responses mediated by high-threshold (i.e. A - and C-fi bre) prim ary aff erent neurons in a manner consistent with an elevated number of nociceptive synapse s in the region (J. Li and Baccei, 2011b). Sensory neurons expressing TRPV1 contrib- ute to this nociceptive synaptic plasticity, as selective destruc- tion of these fi bres by prior systemic capsaicin prevented the injury-evoked increase in spontaneous glutamate release ( J. Li and Baccei, 2011b). In addition, peripheral infl ammation in the neonate, but not adu lt, leads to a proliferation of nociceptive pri- mary aff erent projections to the SDH (Rud a et al., 2000; Walker et al., 2003). While the mechanisms underlying this strengthen- ing of nociceptive synaptic input are not fully understood, NGF signaling at the trkA receptor is likely to play a role (J. Li and Baccei, 2011b). \227\206 Peripheral nociceptors are functional and respond to a range of chemical, thermal and mechanical stimuli. \227\206 Prolonged noxious stimuli or infl ammation produce peripheral sensitization. \227\206 Th e structure and function of spinal cord nociceptive pathways continues to matu re across the fi rst 2 to 3 postnatal weeks in the rodent, a period which may extend throug hout infancy and early childhood in humans. \227\206 Spinal cord signaling is initially characterized by an excess of excitatory eff e cts and delayed development of inhibitory mechanisms. \227\206 Spinal refl ex responses have low thresholds and are poorly local- ized in early life. CHAPTER 6 nociceptive signaling in the periphery and spinal cord 61 Clinical studies evaluating peripheral nociceptive function in neonates and infants show pa rallels with the patterns of refl ex response and changes in sensitivity described. Spinal reflex thresholds are lower in neonates and infants, but a clear relation- ship between the degree of response and intensity of the stimulus is maintained (Andrews and Fitzgerald, 19 99). Sensitization of refl ex responses has clearly been demonstrated following repeated me chanical stimuli (Andrews and Fitzgerald, 1994), repeated heel lance for blood tests (Fitzg erald et al., 1988), and surgery (Andrews and Fitzgerald, 2002) in neonates and infants. Beland, B. and Fitzgerald, M. (2001). Infl uence of peripheral infl ammation on the postnat al maturation of primary sensory neuron phenotype in rats. J Pain , 2 , 36 \200\22345 . Ben-Ari, Y. (2002). Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci , 3 , 728 \200\223739 . Ben-Ari, Y., Cherubini, E., Corradetti, R., and Gaiarsa, J. L. (1989) Giant synaptic potent ials in immature rat CA3 hippocampal neurones. J Physiol , 416 , 303 \200\223325 . Benn, S. C., Costigan, M., Tate, S., Fitzgerald, M., and Woolf, C. J. (2001). Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS 2) in primary sensory neurons. J Neurosci , 21 , 6077 \200\2236085 . An early study evaluating the response of neonates to skin scratch allergy tests demonstrat ed histamine-induced erythema even in preterm neonates (Matheson et al., 1952), and along w ith clinical observations following surgery, suggests that localized infl amma- tory respon ses are functional in early life. Berki, A. C., O \200\231Donovan, M. J., and Antal, M. (1995). Developmental expression of g lycine immunoreactivity and its colocalization with GABA in the embryonic chick lumbosacral spinal cord. J Comp Neurol , 362 , 583 \200\223596 . Berthele, A., Boxall, S. J., Urban, A., Anneser, J. M., Zieglgansberger, W., Urban, L., et al . (1999). Distribution and developmental changes in metabotropic glutamate receptor mess enger RNA expression in the rat lumbar spinal cord. Brain Res Dev Brain Res , 112 , 39 \200 \22353 . Clinical investigations of genetic disorders also demonstrate the importance of nociceptive mechanisms throughout early life. Mutations aff ecting genes encoding the trkA receptor fo r NGF can result in congenital insensitivity to pain and anhidrosis (Indo, 2002). Loss-of-f unction mutations aff ecting Na v 1.7 channels on nociceptive aff erents result in congenit al insensitivity to pain, while gain of function mutations produce episodic severe pain syn dromes (paroxysmal extreme pain disorder or erythromelalgia) that may present in infancy or childhood (Fischer and Waxman, 2010; see also Walker, Chapter 21, this volume). Bhave, G. and Gereau, R. W. (2004). Posttranslational mechanisms of peripheral sensitization. J Neurobiol , 61 , 88 \200\223106 . Bice, T. N. and Beal, J. A. (1997a). Quantitative and neurogenic analysis of the total popu lation and subpopulations of neurons defi ned by axon projection in the superfi cial dorsal horn of the rat lumbar spinal cord. J Comp Neurol , 388 , 550 \200\223564 . Bice, T. N. and Beal, J. A. (1997b). Quantitative and neurogenic analysis of neurons with s upraspinal projections in the superfi cial dorsal horn of the rat lumbar spinal cord. J Com p Neurol , 388 , 565 \200\223574 . Integration of data from laboratory studies is essential to improve our understanding of th e changing impact of pain and injury throughout development, interpret clinical observation s, and ulti- mately improve the management of pain for neonates, infants, and children of a ll ages. Bicknell, H. R., Jr. and Beal, J. A. (1984). Axonal and dendritic development of substantia gelatinosa neurons in the lumbosacral spinal cord of the rat. J Comp Neurol , 226 , 508 \200\223522 . Blankenship, A. G. and Feller, M. B. (2010). Mechanisms underlying spontaneous patterned ac tivity in developing neural circuits. Nat Rev Neurosci , 11 , 18 \200\22329 . References Andrews, K. and Fitzgerald, M. (1994). Th e cutaneous withdrawal refl ex in huma n neonates: sensitization, receptive fi elds, and the eff ects of contralateral stimulation . Pain , 56 , 95 \200\223101 . Boada, M. D., Houle, T. T., Eisenach, J. C., and Ririe, D. G. (2010). Diff ering neurophysi ologic mechanosensory input from glabrous and hairy skin in juvenile rats. J Neurophysiol , 104, 3568 \200\2233575. Boada, M. D. and Woodbury, C. J. (2008). Myelinated skin sensory neurons project extensivel y throughout adult mouse substantia gelatinosa. J Neurosci , 28 , 2006 \200\2232014 . Andrews, K. and Fitzgerald, M. (1999). Cutaneous fl exion refl ex in human neonates: a quan titative study of threshold and stimulus-response characteristics aft er single and repeate d stimuli. Dev Med Child Neurol , 41 , 696 \200\223703 . Bremner, L., Fitzgerald, M., and Baccei, M. (2006). Functional GABA(A)- receptor-mediated i nhibition in the neonatal dorsal horn. J Neurophysiol , 95 , 3893 \200\2233897 . Andrews, K. and Fitzgerald, M. (2002). Wound sensitivity as a measure of analgesic eff ects following surgery in human neonates and infants. Pain , 99 , 185 \200\223195 . Bremner, L. R. and Fitzgerald, M. (2008). Postnatal tuning of cutaneous inhibitory receptive fi elds in the rat. J Physiol , 586 , 1529 \200\2231537 . Andrews, K. A., Desai, D., Dhillon, H. K., Wilcox, D. T., and Fitzgerald, M. (2002). Abdomi nal sensitivity in the fi rst year of life: comparison of infants with and without prenatal ly diagnosed unilateral hydronephrosis. Pain , 100 , 35 \200\22346 . Burnstock, G. (2006). Purinergic P2 receptors as targets for novel analgesics. Pharmacol Th er , 110 , 433 \200\223454 . Charrier, C., Machado, P., Tweedie-Cullen, R. Y., Rutishauser, D., Mansuy, I. M., and Trill er, A. (2010). A crosstalk between beta1 and beta3 integrins controls glycine receptor and gephyrin traffi cking at synapses. Nat Neurosci , 13 , 1388 \200\2231395 . Baba, H., Doubell, T. P., Moore, K. A., and Woolf, C. J. (2000). Silent NMDA receptor-media ted synapses are developmentally regulated in the dorsal horn of the rat spinal cord. J Neu rophysiol , 83 , 955 \200\223962 . Chen, Y., Li, G., and Huang, L. Y. (2012). P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons. Mol Pain , 8 , 9 . Baccei, M. L., Bardoni, R., and Fitzgerald, M. (2003). Development of nociceptive synaptic inputs to the neonatal rat dorsal horn: glutamate release by capsaicin and menthol. J Physi ol , 549, 231 \200\223242. Coggeshall, R. E., Jennings, E. A., and Fitzgerald, M. (1996). Evidence that large myelinat ed primary aff erent fi bers make synaptic contacts in lamina II of neonatal rats. Brain Re s Dev Brain Res , 92 , 81 \200\22390 . Baccei, M. L. and Fitzgerald, M. (2004). Development of GABAergic and glycinergic transmiss ion in the neonatal rat dorsal horn. J Neurosci, 24 , 4749 \200\2234757 . Constantinou, J., Reynolds, M. L., Woolf, C. J., Safi eh-Garabedian, B., and Fitzgerald, M. (1994). Nerve growth factor levels in developing rat skin: upregulation following skin wou nding. Neuroreport , 5 , 2281 \200\2232284 . Cordero-Erausquin, M., Coull, J. A., Boudreau, D., Rolland, M., and De Koninck, Y. (2005) Diff erential maturation of GABA action and ani on reversal potential in spinal lamina I neurons: impact of chloride extrusion capacity. J Neurosci , 25 , 9613 \200\2239623 . Bardoni, R., Magherini, P. C., and MacDermott, A. B. (1998). NMDA EPSCs at glutamatergic sy napses in the spinal cord dorsal horn of the postnatal rat. J Neurosci , 18 , 6558 \200\223 6567 . Beggs, S., Torsney, C., Drew, L. J., and Fitzgerald, M. (2002). Th e postnatal reorganizati on of primary aff erent input and dorsal horn cell receptive fi elds in the rat spinal cord is an activity-dependent process. Eur J Neurosci , 16 , 1249 \200\2231258 . SECTION 2 biological basis of pediatric pain 62 Goldstein, S. A., Bockenhauer, D., O \200\231Kelly, I., and Zilberberg, N. (2001). Potassiu m leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci , 2 , 175 \200\223184 . Coull, J. A., Boudreau, D., Bachand, K., Prescott, S. A., Nault, F., Sik, A., et al . (2003 ). Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neur opathic pain. Nature , 424 , 938 \200\223942 . Granmo, M., Petersson, P., and Schouenborg, J. (2008). Action-based body maps in the spinal cord emerge from a transitory fl oating organization. J Neurosci , 28 , 5494 \200\2235503 . Daniele, C. A. and MacDermott, A. B. (2009). Low-threshold primary aff erent drive onto GAB Aergic interneurons in the superfi cial dorsal horn of the mouse. J Neurosci , 29 , 686 \200\223695 . Green, G. M. and Gibb, A. J. (2001). Characterization of the single-channel properties of N MDA receptors in laminae I and II of the dorsal horn of neonatal rat spinal cord. Eur J Neu rosci , 14 , 1590 \200\2231602 . Davidson, S., Truong, H., and Giesler, G. J. Jr. (2010). Quantitative analysis of spinothal amic tract neurons in adult and developing mouse. J Comp Neurol , 518 , 3193 \200\2233204 . Guo, A., Simone, D. A., Stone, L. S., Fairbanks, C. A., Wang, J., and Elde, R. (2001). Deve lopmental shift of vanilloid receptor 1 (VR1 ) terminals into deeper regions of the superfi cial dorsal horn: correlation with a shift from TrkA to Ret expression by dorsal root gang lion neurons. Eur J Neurosci , 14 , 293 \200\223304 . Delmas, P., Hao, J., and Rodat-Despoix, L. (2011). Molecular mechanisms of mechanotransduct ion in mammalian sensory neurons. Nat Rev Neurosci , 12 , 139 \200\223153 . Dib-Hajj, S. D., Cummins, T. R., Black, J. A., and Waxman, S. G. (2010). Sodium channels in normal and pathological pain. Annu Rev Neurosci , 33 , 325 \200\223347 . Guy, E. R. and Abbott, F. V. (1992). Th e behavioral response to formalin in preweanling rats. Pain , 51 , 81 \200\22390 . Dougherty, K. J., Sawchuk, M. A., and Hochman, S. (2009). Phenotypic diversity and expressi on of GABAergic inhibitory interneurons during postnatal development in lumbar spinal cord of glutamic acid decarboxylase 67-green fl uorescent protein mice. Neuroscience , 163 , 909 \200\223919 . Hartmann, B., Ahmadi, S., Heppenstall, P. A., Lewin, G. R., Schott, C., Borchardt, T., et a l . (2004) Th e AMPA receptor subunits GluR-A and GluR-B reciprocally modulate spinal synap tic plasticity and infl ammatory pain. Neuron , 44 , 637 \200\223650 . Harvey, R. J., Depner, U. B., Wassle, H., Ahmadi, S., Heindl, C., Reinold, H., et al . (200 4). GlyR alpha3: an essential target for spinal PGE2-mediated infl ammatory pain sensitizat ion. Science , 304 , 884 \200\223887 . Ekholm, J. (1967). Postnatal changes in cutaneous refl exes and in the discharge pattern of cutaneous and articular sense organs. A morphological and physiological study in the cat. Acta Physiol Scand Suppl , 297 , 1 \200\223130 . Fabrizi, L., Slater, R., Worley, A., Meek, J., Boyd, S., Olhede, S., et al . (2011). A shift in sensory processing that enables the d eveloping human brain to discriminate touch from pain. Curr Biol , 21 , 1552 \200\2231558 .