Development of Pain Pathways and Mechanisms

Early Specification of Nociceptors

Pain development begins with the emergence of nociceptors as a distinct subpopulation of sensory neurons, separate from low-threshold mechanoreceptors and proprioceptors. The majority of sensory neurons originate from neural crest cells and become specified as nociceptors, thermoreceptors, mechanoreceptors, or proprioceptors in the dorsal root ganglia (DRGs) ( ). Both the survival and phenotype of sensory neurons depend on signaling from distinct neurotrophin receptors belonging to the Trk (tyrosine kinase) family ( ). Beginning at embryonic day 11 (E11) in the rat, axons from DRGs innervate the periphery in an organized proximal-to-distal manner such that by birth, sensory fibers have reached the most distal skin of the foot ( ). Sensory fibers grow to peripheral and central targets with remarkable precision, and from the outset each DRG innervates characteristic skin dermatomes ( ). Target innervation critically depends on both nerve growth factor (NGF) signaling and activation of the serum response factor (SRF) transcription factor ( ), as well as the interaction of Trk receptors with leucine-rich repeat and immunoglobulin (LIG) family proteins ( ). The overall number of DRG neurons does not change significantly after birth, but neurotrophin support continues to be required for the first postnatal week in rodents, as shown by the dramatic fall after neonatal axotomy ( ).

The generation of small-diameter nociceptive neurons is dependent on the interaction between intrinsic transcription factors expressed by the developing neurons and external, target-derived signals, which together specify functionally distinct sensory neurons for pain, temperature, and itch (Lui and Ma 2011). The initial specification of the broad category of small-diameter C-fiber nociceptors depends on the expression of neurogenin 1 (Ngn1) transcription factors in early embryonic life ( ). These neurons initially all express TrkA and require NGF for survival but, later in embryonic development, are divided into peptidergic and non-peptidergic classes under the influence of the transcription factor Runx1 ( Fig. 9-2 ). Thus, a subset of these neurons down-regulate TrkA during early postnatal life and become dependent on glial cell line–derived neurotrophic factor (GDNF) via expression of the GDNF receptor Ret, which can modulate the mechanical sensitivity of these nociceptors ( , ). In this way, the Ret-expressing, isolectin B4 (IB4)-positive, non-peptidergic phenotype emerges as a distinct group of nociceptors only postnatally. This switch to Ret signaling is driven by NGF itself, which also initiates expression of the transcription factor Runx1 ( ) and thereby leads to the extinction of calcitonin gene–related peptide (CGRP) expression and promotion of the non-peptidergic phenotype ( ). Meanwhile, Runx1 activity is suppressed in peptidergic neurons via hepatocyte growth factor (HGF)-Met signaling ( ). Recent evidence suggests that brain-derived neurotrophic factor (BDNF) signaling during the neonatal period is also required for the survival of both peptidergic and non-peptidergic nociceptors ( ). Figure 9-2 summarizes these events.

The generation of TrkC-expressing proprioceptors and cutaneous mechanoreceptors (expressing TrkB) can be distinguished from nociceptors by their dependence on the Ngn2 transcription factor ( ). Within the Ngn2 population, the activity of transcription factor Runx3 promotes the proprioceptive identity ( ) by decreasing TrkB expression in a subset of TrkC-expressing neurons ( ). Although many functional subtypes of low-threshold mechanoreceptors have been characterized, recent evidence shows that afferents innervating Merkel cells, hair follicles, and Meissner corpuscles all originate from a subpopulation of embryonic DRG neurons that co-express the transcription factor MafA with the GDNF receptors Ret and glial cell line-derived neruotrophic factor family receptor (GFR)-α2 shortly after genesis within the DRG ( ). These “early” Ret-expressing neurons constitute a separate population from the non-peptidergic nociceptors that express Ret later in life ( ).

Maturation of Nociceptor Transduction and Transmission

Nociception begins with the transduction of noxious mechanical, thermal, or chemical stimuli through a set of specific transient receptor potential (TRP) channels expressed in nociceptor terminals. TRP expression is detected in embryonic life ( ), but with some notable subtype differences. The capsaicin receptor TRPV1, which responds to heat, protons, toxins, and the chilli pepper ingredient capsaicin, is expressed by E12.5 in the mouse, whereas TRPA1 channels, which respond to chemical irritants and noxious cold, are not expressed in nociceptors in the first weeks of life ( ), which explains the weak nociceptive reflex responses to the TRPA1 agonist mustard oil (allyl isothiocyanate) in young animals ( ). Conversely, expression of adenosine triphosphate (ATP)-gated P2X ₃ ion channels undergoes a postnatal switch from all DRG cells to IB4-positive neurons only, which explains the postnatal reduction in electrophysiological and behavioral sensitivity to P2X receptor (P2XR) agonists (Chen et al 2012). Control of the expression patterns of sensory receptors and channels during development is an area of rapidly changing research. Runx1, which is initially expressed in most TrkA-positive neurons, together with two other transcription factors, islet1 and Brn3a, coordinates the expression of a variety of sensory channels and receptors, including P2X ₃ , TRPA1, TRPM8, the Mrgpr family of G protein–coupled receptors (GPCRs), the sodium channel Na _v 1.9, and the high TRPV1 expression observed in a small subset of DRG neurons ( ).

Functional polymodal nociceptors can be identified in fetal and neonatal life ( ) by their distinctive patterns of spike activity in response to a range of cutaneous noxious stimulation, including some activity that is not normally observed in adults ( , ). Nociceptors can be clearly distinguished from low-threshold mechanoreceptors in the fetal rat ( ), but the stimulus–response characteristics of the latter continue to mature over postnatal life ( , , ). The ability of unmyelinated nociceptors to respond to noxious heat depends on NGF during the early postnatal period inasmuch as treatment with NGF antisera between postnatal day 2 (P2) and P14 converts C-fiber mechano-heat receptors to a novel type of low-threshold mechanical pressure receptor ( ) whereas systemic neonatal NGF treatment leads to an increase in the proportion of C-fiber heat nociceptors ( ). Meanwhile, the number of cutaneous A-fiber mechano-heat nociceptors decreases during the first 10 postnatal days ( ), although whether this relates to postnatal changes in neurotrophin signaling remains unclear. Conduction velocities in immature afferent sensory nerves are lower than in mature nerves because the axons are smaller and incompletely myelinated. As a result, nociceptive transmission through Aδ- and C-fiber axons and innocuous sensory transmission through Aβ axons is slower in young mammals, and the frequency of action potential (AP) firing is also lower because of immature ion channel properties, which leads to less reliable stimulus frequency coding. The tetrodotoxin-resistant sodium channel Na _v 1.8 (SNS/PN3) is expressed in developing C-fiber neurons by E17, with adult levels observed by P7 ( ).

Early Development of Dorsal Horn Neurons

Complete understanding of nociceptive processing in the immature nervous system requires characterization of the ontogeny of spinal networks in the dorsal horn. The spinal cord develops along a ventrodorsal gradient such that deep dorsal horn neurons are born after motoneurons and substantia gelatinosa (SG) cells are the last to mature ( ). Within the SG, supraspinal lamina I projection neuron generation is complete before that of propriospinal neurons and local circuit interneurons ( ). The specification of neurotransmitter phenotype within these interneurons is under tight regulatory control from an extensive complement of homeodomain (HD) and basic helix–loop–helix (bHLH) transcription factors.

From E11.5, two late-born populations of dorsal horn neurons populate the superficial laminae and are distinguished by their complementary expression of the HD genes Lmx1b and Pax2 ( ). Pax2 , along with Lbx1 , is required to induce the differentiation of both GABAergic ( ) and glycinergic ( ) interneurons within the developing dorsal horn. The specification of glutamatergic interneurons in the embryonic dorsal horn results from active repression of the GABAergic phenotype by the HD transcription factors Tlx3 and Tlx1 ( ). Recent work also suggests a role of the transcription factor DRG11 (i.e., Prrxl1 ) in promoting the glutamatergic phenotype within the dorsal horn ( ). Thus, the final proportions of GABAergic and glutamatergic interneurons in the embryonic dorsal spinal cord appear to be determined by the balance between Tlx3 and Lbx1/Pax2 expression ( ). This balance is also instrumental in the generation of dorsal horn neurons that express neuropeptides since cholecystokinin- and substance P (SP)-expressing neurons depend on Tlx3 and Tlx1 ( ) whereas cells containing enkephalin, neuropeptide Y, or galanin appear to require the expression of Ptf1a , Lbx1 , and Pax2 ( ).

Interneurons within the superficial dorsal horn (SDH) undergo significant morphological changes during the early postnatal period ( ) that depend in part on calcium-calmodulin–dependent kinase IIα (CAMKIIα) signaling ( ). Despite this growth of dendritic and axonal processes in the neonate, the thickness of laminae I and II _outer remains relatively constant from birth to adulthood ( ). SDH neurons also exhibit developmental changes in their intrinsic membrane properties during early life, including up-regulation of A-type K ⁺ currents and an increase in AP amplitude ( ). The patterns of AP discharge seen in SDH neurons shift with age, with a greater proportion of cells firing repetitively in response to intracellular current injection ( ). In addition, the neonatal SDH is distinguished by the presence of “pacemaker” neurons within lamina I, which are defined by their intrinsic ability to generate spontaneous, rhythmic burst firing ( ). Although this pacemaker activity appears to be confined to interneurons during early life, both spino-parabrachial and spino–periaqueductal gray (PAG) lamina I projection neurons exhibit spontaneous activity throughout postnatal development ( ).

Growth of Mechanoreceptor A-Fiber Afferents into the Dorsal Horn

Large-diameter myelinated A-fiber afferents are the first to grow into the dorsal horn at E15 in the rat and from 11 weeks in humans ( , , ). The initial A-fiber terminal fields are diffusely distributed in the dorsoventral and rostrocaudal dimensions, gradually reorganize during the early postnatal period, and exhibit a nearly adult pattern by P21 ( , ). The more dorsal termination of A fibers in the first postnatal weeks means that they overlap in the SDH with C-fiber terminals. Not all A-fiber terminal arbors do this, but those that do appear to form synaptic contacts in lamina II ( , , ). The functional class of afferent that gives rise to these exuberant terminals is not clear, but they may arise partly from myelinated high-threshold mechanoreceptors ( ). Nonetheless, a high incidence of Aβ-evoked monosynaptic responses has been observed in patch-clamp studies of immature SG neurons ( , ), and polysynaptic Aβ-fiber synapses onto GABAergic neurons in laminae I–II are more prevalent in neonates ( ). In addition, activation of Aβ fibers evokes c-fos expression in the SG at P3 ( ), and repetitive A-fiber stimulation can produce sensitization in dorsal horn neurons in neonates but not in adults ( ).

Therefore, neurons in the SDH receive a greater degree of Aβ-mediated input during the neonatal period, either from direct Aβ-fiber projections to the region or from low-threshold, polysynaptic connections.

Growth of Nociceptor C-Fiber Afferents into the Dorsal Horn

Neonatal pain processing requires the formation of central connections between nociceptive primary afferents and spinal cord neurons. Nociceptive projections to the dorsal horn are not observed until E19 in the rodent ( ) and are dependent on activation of the paired HD transcription factor DRG11 ( ). From the outset, these fibers terminate in a somatotopically precise manner in laminae I–II of the dorsal horn ( ) and include SP, CGRP, galanin, and somatostatin-expressing fibers ( , ). The timing of C-fiber growth into the human fetal spinal cord remains unclear, but no afferent terminals were observed in lamina II at 19 weeks by DiI labeling ( ). Despite the anatomical presence of C fibers in the rodent spinal cord during the embryonic period, evidence points to delayed maturation of C-fiber synaptic connections in the SDH during the early postnatal period. Extracellular single-unit recordings in vivo have demonstrated that although low-intensity electrical stimulation of A fibers evokes APs in dorsal horn neurons from P3, long-latency C-fiber–evoked activity is not apparent until P10 ( ). This is not due to an absence of functional synapses before birth since electron microscopic studies have demonstrated the existence of synaptic terminals corresponding to small-diameter sensory afferents from birth ( ). In addition, capsaicin application increased release of glutamate in the SDH from P0, thus suggesting that nociceptive afferents expressing TRPV1 begin forming functional synapses before birth. During the neonatal period, the number of C-fiber synapses may be insufficient to effectively drive suprathreshold membrane depolarization in SDH neurons, whereas a subsequent proliferation of C-fiber synaptic input between P5 and P10 ( ) may underlie the appearance of C-fiber–evoked spiking at later ages.

Maturation of Excitatory Synaptic Transmission in the Developing Dorsal Horn

Inhibitory Synaptic Transmission in the Developing Dorsal Horn

Nociceptive processing requires effective inhibitory as well as excitatory transmission within the dorsal horn through the two major neurotransmitters, γ-aminobutyric acid (GABA) and glycine ( Fig. 9-3 ).