Development and terminal differentiation of pulp and periodontal nerve elements in subcutaneous transplants of molar tooth germs and incisors of the rat.

Ectopic tooth transplants are known to receive rich innervation of local neurons, but the precise location and structural features of neurites in the pulp and periodontal ligament (PDL) of such transplants are unclear. In this experiment, the molar tooth germs of rat embryos and incisors of young rats were subcutaneously transplanted into the dorsal regions of rats and processed, at various time intervals, for immunohistochemical demonstration of neural elements. Teeth with periodontal tissue elements developed in most of the molar transplants in 6 or 8 wk and received rich innervation, including some autonomic fibres, in the pulp. Nerve elements were also confirmed to be present in the PDL of these transplants, including specialized nerve ending-like structures reminiscent of the periodontal Ruffini endings. Mechanoreceptor-like structures were also induced in the regenerated PDL of similarly transplanted incisors, although the success rate was low. We conclude that rich and highly ordered innervation of the pulp, and occasional development of mechanoreceptors in the regenerated PDL of ectopic dental transplants, imply a high probability of successful induction of teeth with both nociceptive and mechanical sensations in the ectopic tooth and/or tooth germ transplant systems, although differentiation of mechanoreceptor-like nerve endings occurred in only a few rare cases.

Postnatal changes in the Rexed lamination and markers of nociceptive afferents in the superficial dorsal horn of the rat.

In this study, we investigated postnatal changes in Rexed's laminae and distribution of nociceptive afferents in the dorsal horn of the rat lumbar spinal cord at postnatal days 0, 5, 10, 15, 20, and 60. Transverse sections of the L4-L5 segments were processed for triple labeling with isolectin B4 (IB4)-binding as a marker of nonpeptidergic C-fibers, calcitonin gene-related peptide (CGRP) immunoreactivity to label peptidergic nociceptive afferents, and a fluorescent Nissl stain to visualize cells and lamination at different stages of postnatal development. The Nissl staining revealed that the thickness of lamina I (LI) and outer lamina II remained mostly unchanged from birth until adulthood. CGRP afferents terminated mostly in LI and the outer two-thirds of lamina II, whereas the termination area of fibers binding IB4 was centered on the middle one-third of lamina II at all ages studied. In absolute values, the overall width of the bands of intense CGRP and IB4 labeling increased with age but decreased as a percentage of the overall thickness of the dorsal horn with maturation. The overlap of CGRP termination area with that of IB4 afferents increased with age. The consequences of these findings are twofold. First, the size of the different laminae does not grow evenly across the dorsal horn. Second, CGRP and IB4 labeling cannot be considered per se to be reliable markers of lamination during development. These findings have implications for comparing data obtained in immature and mature tissues with respect to localization of structures in the dorsal horn.

Hierarchical control of sensory neuron development by neurotrophic factors.

We are still far from reaching a complete understanding of the molecular mechanisms that control neuronal diversification during nervous system development. In this issue of Neuron, Luo et al. bring that goal a step closer by revealing how a hierarchical interaction between two neurotrophic factor systems drives the differentiation, maturation, and extension of peripheral projections in a subclass of sensory neurons that mediate pain perception.

Target finding of pain nerve fibers: neural growth mechanisms in the tooth pulp.

The tooth pulp has a dense sensory innervation which, upon stimulation, conveys sensory signals perceived as pain. This innervation, which originates from the trigeminal ganglion, is established through a series of regulated steps during development, and represents an interesting example of tissue targeting by pain-specific nerves. We have investigated various potentially neurotrophic and neurorepulsive influences during this process. The dental papilla/pulp appears to secrete neurite growth inhibitory molecular factors at early stages, which prevent nerve fibers from entering the tissue at what appears to be inappropriate timepoints. Later, a shift from repulsive to attractive factors apparently takes place, and nerve fibers then enter the tooth. When nerve fibers have invaded the dental mesenchyme, a complicated interplay of secreted and membrane-bound factors probably directs the nerve terminals to appropriate sites. Laminin-8 (alpha4beta1gamma1, Lm-411), which is produced by pulpal cells, emerges as an important candidate molecule in this context. Insights into the interactions between the dental pulp nerve fibers and their environment may become important in the search for novel ways to ameliorate pain in the tooth, as well as at other sites.

[Neurobiology of the chronicisation of pain in children: the memory of pain and its painful memory]. / La neurobiologie de la chronicisation de la douleur chez l'enfant: la mémoire de la douleur et la douleur de la mémoire.

Reviewing the development of nociceptive circuits provides the rationale behind the need to modify and reduce premature painful experiences, especially during the "plastic" neonatal phase. Indeed, if physiological mechanisms of the functional nociceptive system follow a harmonious and predetermined development, it is the individual personal experience, intrinsically random, which will shape the final reactivity of this system and the later painful experience. If pain would not have been the organism's alarm system, we could have simply compared it by analogy to other sensorial systems, which its development depends exclusively on the presence of environmental stimuli. The eyes wait for light, the ears for sound, the skin to be touched, the tongue to taste and the olfactory bulbs to smell. However with pain it is not the quantitative exposure that determines its development, but rather the context-laden aspects of its affliction which in turn create the complex experience and "memory" of pain. Prolonged, but also "unnecessary" exposure to pain transforms it into a futile sensation, which impacts the individual immediately but also resonates into its future. This article reviews recent neurobiological mechanisms (such as neural circuitry, neurotrophins, peripheral and central sensitization, inhibitory pathways) now known to develop during the chronicisation and apprenticing of pain in the growing individual. Its cognizance is vital for a better comprehension of adult pain.

Percepción del dolor en los niños. Analgesia multimodal perioperatoria / Pain perception in children. Various peri-operative analgesia modes

Tradicionalmente, el dolor en el niño se ha tratado de forma insuficiente. Los neo0natos a menudo deben someterse a intervenciones invasivas, y actualmente existen suficientes datos para afirmar que ellos son capaces de percibir el dolor. El tratamiento incompleto del dolor conlleva un aumento de la morbimortalidad. En los últimos años se han producido numerosos avances en el cuidado y manejo del recién nacido. Existen diversas escalas de medida para la evaluación del dolor en neonatos a término y pretérmino. Ellas se basan en la observación y registro de las alteraciones fisiológicas o en cambios del comportamiento, o una combinación de ambos. En este artículo se hace un repaso de las más utilizadas. Hay una serie de medidas generales de vital importancia en el tratamiento del dolor en neonatos, como evitar procedimientos dolorosos innecesarios, el cuidado del ambiente que le rodea, la administración de distintos tipos de soluciones orales dulces, la estimulación multisensorial y el amamantamiento materno durante la intervención dolorosa. Sin embargo, en ciertas ocasiones estos recursos son insuficientes y debemos recurrir a las medidas farmacológicas. Los fármacos más utilizados son los anestésicos locales, los opioides y los analgésicos antinflamatorios no esteroideos.

Percepción del dolor en los niños. Analgesia multimodal perioperatoria / Pain perception in children. Various peri-operative analgesia modes

Tradicionalmente, el dolor en el niño se ha tratado de forma insuficiente. Los neo0natos a menudo deben someterse a intervenciones invasivas, y actualmente existen suficientes datos para afirmar que ellos son capaces de percibir el dolor. El tratamiento incompleto del dolor conlleva un aumento de la morbimortalidad. En los últimos años se han producido numerosos avances en el cuidado y manejo del recién nacido. Existen diversas escalas de medida para la evaluación del dolor en neonatos a término y pretérmino. Ellas se basan en la observación y registro de las alteraciones fisiológicas o en cambios del comportamiento, o una combinación de ambos. En este artículo se hace un repaso de las más utilizadas. Hay una serie de medidas generales de vital importancia en el tratamiento del dolor en neonatos, como evitar procedimientos dolorosos innecesarios, el cuidado del ambiente que le rodea, la administración de distintos tipos de soluciones orales dulces, la estimulación multisensorial y el amamantamiento materno durante la intervención dolorosa. Sin embargo, en ciertas ocasiones estos recursos son insuficientes y debemos recurrir a las medidas farmacológicas. Los fármacos más utilizados son los anestésicos locales, los opioides y los analgésicos antinflamatorios no esteroideos. (AU)

Opioid analgesia in the newborn.

Pain in neonates is now well established. Studies of the developmental neurobiology of pain have revealed that pain processing in the immature is very different from that in the mature nervous system. Neonates undergo considerable maturation of peripheral, spinal and supraspinal afferent pain transmission over the early postnatal period but are able to respond to tissue injury with specific behaviour and with autonomic, hormonal and metabolic signs of stress and distress. Opioid analgesia is now widely used in neonates. There is evidence that morphine requirements may be low in the youngest patients. Sensory threshold testing in rat pups has shown that the analgesic potency of systemic morphine mechanical stimulation is significantly greater in the neonate and declines with postnatal age. The changing morphine sensitivity in the postnatal period may be part of a general reorganisation in the structure and function of primary afferent synapses, neurotransmitter/receptor expression and function and excitatory and inhibitory modulation from higher brain centres. Importantly opioid receptor expression undergoes significant developmental regulation - mu opioid receptors, observed to be exuberantly expressed in the neonatal rat, have been found to be functional. These findings have important implications for the human neonate as they provide a possible explanation for the differences in morphine requirements observed in the youngest patients. The study of the underlying mechanisms of pain and analgesia in development has enabled important changes in clinical practice. However, pain in the newborn remains poorly understood and continued research and intensive study in this area is essential for further effective analgesic intervention and the discovery of new targets for therapy.

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

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

Rescue of NGF-deficient mice I: transgenic expression of NGF in skin rescues mice lacking endogenous NGF.

Mice lacking a functional NGF gene (ngf-/- mice) have less than one third of the normal complement of sensory neurons, few sympathetic postganglionic neurons and die shortly after birth. We report here that transgenic expression of NGF under control of the K14 keratin promoter can rescue some elements of the peripheral nervous system and restore normal growth and viability to ngf-/- mice. While hybrid transgenic-ngf-/- mice (ngfTKOs) displayed marginal rescue of trigeminal ganglion neurons, the percentage of CGRP-positive neurons was restored to normal. Restoration of CGRP-positive terminals in skin and spinal cord was also found and accompanied by recovery of behavioral responses to noxious stimuli. ngfTKO mice displayed a normal number of superior cervical ganglion neurons and recovery of sympathetic innervation of skin. These results demonstrate that substitution of a functional NGF locus by a transgene directing expression largely to skin can result in normal growth and viability. Thus, the most vital functions of NGF are not dependent on faithful recapitulation of the normal spatiotemporal pattern of gene expression.

The postnatal development of tail-flick latencies to acute and repeated stimulation in the rat.

The development of the reaction to thermal nociceptive stimulation was studied in neonatal rats of two strains (albino Wistar and pigmented Long-Evans). Using the tail-flick method in neonates of both strains the effects of daily repeated thermal stimulation (sensitisation) were compared to those of acute stimulation applied on 1 day only. During the whole postnatal development period studied (3-28 days) the pigmented rat pups were more sensitive to a repeated thermal nociceptive stimulation than the albinos. The latency of the response did not change linearly during the developmental time studied, but had a pronounced peak (decreased sensitivity) in the period of eye-lid opening. In the pigmented animals the peak appeared 1 day earlier than in the albinos. At the time of the weaning period the latencies of the response were again shorter.

Maturational changes in the thermoalgesic system in humans from childhood to adulthood revealed by CO(2) laser evoked brain potentials following cutaneous heat stimuli.

Possible maturational changes in the thermoalgesic system were studied by reaction times (RT) and late (Adelta-fibre) laser evoked potentials (LEPs) following CO(2) laser heat stimulation of the hand in healthy children (n=12) and young adults (n=12). In children (10+/-2 years) LEPs presented a negative-positive complex with maximum amplitude (peak-to-peak 71+/-35 microV) at the vertex and latencies of 248+/-82 and 433+/-104 ms, respectively. As compared to adults (24+/-3 years), children had a significant increased peak-to-peak amplitude (+25.7 microV; P=0.03) although no difference in latencies and topography. Median RT (710 ms) was also significantly increased (+312 ms; P<0.005) in children. A decrease in RT and late LEP amplitude from childhood to adulthood may reflect aspects of maturation in sensory processing of the thermoalgesic system.

Altered nociceptive response in mice deficient in the alpha(1B) subunit of the voltage-dependent calcium channel.

Calcium influx through N-type calcium channels mediates synaptic transmission at numerous central synapses and transduces nociceptive information in the spinal dorsal horn. However, the precise role of N-type calcium channels in pain perception is not fully elucidated. To address this issue, we generated and analyzed knockout mice for alpha(1B,) the pore-forming subunit of the N-type calcium channel. Homozygous mutants are viable, fertile, and show normal motor coordination. In small-diameter dorsal root ganglion neurons from mutants the density of calcium channel currents is significantly reduced, which can be accounted for by the abolition of N-type currents. We performed several pain-related behavioral tests using the mutant mice. alpha(1B)-Deficient mice show reduced response to mechanical stimuli in the von Frey test and increased tail flick latency in response to radiant heat, indicating altered spinal reflexes. However, pain response in the hot plate test is normal. In the formalin paw test, the mutant mice exhibit significantly attenuated response in Phase 2, but normal pain behaviors in Phase 1. The response to visceral inflammatory pain caused by acetic acid is also reduced in alpha(1B) knockout mice. These results suggest that the alpha(1B) subunit of N-type calcium channel plays a major role in pain perception by acting at the spinal level, but not at the supraspinal level.

Characterization of the single-channel properties of NMDA receptors in laminae I and II of the dorsal horn of neonatal rat spinal cord.

The single-channel properties of native NMDA receptors in laminae I and II of the dorsal horn of the neonatal rat spinal cord were studied using outside-out patch-clamp techniques. These receptors were found to have several features that distinguish them from native NMDA receptors elsewhere in the CNS. Single-channel currents activated by NMDA (100 nm) and glycine (10 microm) exhibited five distinct amplitude components with slope-conductance values of 19.9 +/- 0.8, 32.9 +/- 0.6, 42.2 +/- 1.1, 53.0 +/- 1.0 and 68.7 +/- 1.5 pS. Direct transitions were observed between all conductance levels but transitions between 69-pS openings and 20-, 33- and 42-pS openings were rare. There was no significant difference in the frequency of direct transitions from 42- to 20-pS compared to 20- to 42-pS transitions. The Kb (0 mV) for Mg2+ was 89 microm. The Mg2+ unblocking rate constant was similar to other reported values. However, the Mg2+ blocking rate constant was larger than other reported values, suggesting an unusually high sensitivity to Mg2+. The NR2B subunit-selective antagonist, ifenprodil, had no significant effect on overall channel activity but significantly decreased the mean open time of 53-pS openings. These results suggest neonatal laminae I and II NMDA receptors are not simply composed of NR1 and NR2B subunits or NR1 and NR2D subunits. It is possible that these properties are due to an as yet uninvestigated combination of two NR2 subunits with the NR1 subunit or a combination of NR3A, NR2 and NR1 subunits.

Postnatal development of central nociceptive mechanisms modulating jaw muscle activity in the rat.

Postnatal changes in the electromyographic (EMG) activity of jaw muscles evoked by mustard oil (MO) application into the rat temporomandibular joint region and the recurrence of increased jaw muscle activities after intravenous injection of naloxone were compared among 4, 6 and 8-week-old rats. In all the groups, MO application increased EMG activity on the ipsilateral side, however, 4-week old rats showed only a small increase in EMG activity on the contralateral side. The EMG activity on the contralateral side increased in an age-dependent manner. The recurrence of increased jaw muscle activity was not induced in 4-week old rats. These results suggest that a neural circuit for generating contralateral responses and mechanisms for central excitation are not established until after 4 weeks postnatally.

Postnatal development of the nociceptive withdrawal reflexes in the rat: a behavioural and electromyographic study.

1. The postnatal development of nociceptive withdrawal reflexes was studied. In awake intact rats, forelimb, hindlimb and tail reflexes were recorded on videotape. In decerebrate spinal rats, electromyography (EMG) was used to record nociceptive withdrawal reflexes in musculi extensor digitorum longus (EDL), peronei, gastrocnemius-soleus (G-S) and biceps posterior-semitendinosus (BP-ST). Thermal (short-lasting CO2 laser pulses) and mechanical stimulation were used. 2. In adults, nociceptive withdrawal reflexes were typically well directed and reflex pathways to single hindlimb muscles had functionally adapted receptive fields. By contrast, at postnatal day (P) 1-7, the nociceptive withdrawal reflexes were often inappropriate, sometimes producing movements towards the stimulation, and EMG recordings revealed unadapted variable receptive fields. With increasing age, the nociceptive withdrawal reflexes progressively became well directed, thus producing localized withdrawal. Both withdrawal movements and spatial organization of the receptive fields were adult-like at P20-25. 3. Up to P25, reflex thresholds were more or less constant in both intact awake rats and spinal decerebrate rats, except in G-S in which no nociceptive withdrawal reflexes were evoked from P20 on. After P25, mechanical, but not thermal, thresholds increased dramatically. 4. EMG recordings revealed that during the first three postnatal weeks, the latency of the CO2 laser-evoked nociceptive withdrawal reflexes decreased significantly in peronei and BP-ST, but not in EDL, and thereafter increased significantly in peronei, BP-ST and EDL. The magnitude of the nociceptive withdrawal reflexes in these muscles increased markedly between P7 and P20 and showed little change thereafter. 5. Possible mechanisms underlying the postnatal tuning of the nociceptive withdrawal reflexes are discussed.

GAP-43 in the spinal trigeminal and dorsal column nuclei of the newborn and adult man: immunohistochemical distribution and comparison with that of the neuropeptides SP and CGRP.

By means of immunohistochemistry the presence of the growth-associated protein GAP-43 and its codistribution with substance P (SP) and calcitonin gene-related peptide (CGRP) are studied in the human spinal trigeminal, gracile, and cuneate nuclei at perinatal and adult life stages. The results obtained show that the distribution pattern of GAP-43 in the areas examined varies with age and that the immunohistochemical detectability of the protein persists in discrete subregions of the trigeminal and cuneate nuclei of the adult, where its localization closely matches that of SP and CGRP. It is suggested that neuronal plasticity may be pronounced throughout life in areas of the human nervous system involved in the neurotransmission of protopathic stimuli at the first synaptic level. Discrete subregions of the cuneate nucleus, bearing neurochemical characteristics strikingly similar to those of the substantia gelatinosa of the trigeminal subnucleus caudalis are pointed out.

Effect of chronic maternal diazepam treatment on the development of stress-induced antinociception in young rats.

The use and abuse of benzodiazepines is widespread and we have begun to address whether maternal exposure to these drugs influences the development of opioid systems. We have studied the effect of maternal diazepam treatment on stress-induced antinociception in the neonatal offspring. Diazepam (1 or 10 mg/kg) was administered twice daily to mothers from conception. Pre- and postweanling rat pups were assessed for opioid-mediated stress-induced antinociception by 3-min swimming and measuring nociception using the tail immersion test. In preweanling rats there was stress-induced antinociception in both vehicle- and diazepam-treated animals but in diazepam-treated groups (1 and 10 mg/kg) this was insensitive to reversal by the opioid antagonist naloxone, suggesting that nonopioid systems are operating this response. In postweanling rats a similar insensitivity to naloxone was observed in 1 mg/kg diazepam-treated groups; with 10 mg/kg diazepam there was no significant antinociception. The results suggest that maternal diazepam treatment interferes with the development of stress-mediated responses and that part of this toxicity is due to actions on opioid systems in the CNS.

Requirement for nerve growth factor in the development of myelinated nociceptors in vivo.

In adult animals, sensory neurons innervating the skin are phenotypically diverse. We have now investigated whether nerve growth factor (NGF) has a physiological role in the development of this diversity. We gave antisera against NGF to rats from postnatal day 1 (PND 1) to adulthood (5 weeks). We found a virtually complete depletion of high threshold mechanoreceptors conducting in the A delta range (2-13 ms-1) in the sural nerve. This afferent type, normally present in large numbers, appeared to have been replaced by D-hair afferents, sensitive mechanoreceptors which normally are relatively rare. NGF deprivation had this effect only in early postnatal life; treatment from postnatal day 14 to adulthood had no effect. We conclude that the presence of NGF postnatally in skin is necessary for the proper phenotypic development of A delta cutaneous nociceptors.