DOI 10.1007/s11055-015-0094-8 Neuroscience and Behavioral Physiology, Vol. 45, No. 4, May, 2015
Neurochemical Characteristics of Sensory Neurons during Ontogeny P. M. Maslyukov,1 V. V. Porseva,1 M. B. Korzina,1 and A. D. Nozdrachev2
Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 99, No. 7, pp. 777–792, July, 2013. Original article submitted June 10, 2013. Sensory neurons constitute a heterogeneous population of neurons with different morphological, receptor, and immunohistochemical characteristics. Most large neurons with myelinated fibers of the Aδ group contain 200-kDal neurofilament protein (NF200), while some small afferent intervertebral ganglion neurons can bind isolectin B4 (IB4). Sensory neurons can contain different types of tyrosine kinase (A, B, and C) and have different neurotransmitter compositions. Neuropeptides are found mainly in neurons of small and intermediate size. The proportion of neurons containing tyrosine kinase A decreases and the proportions of neurons positive for NF200, IB4, substance P, and CGRP increase during early postnatal ontogeny. The growth and development of sensory neurons, especially during the embryonic period, occurs under the influence of neurotrophins. Keywords: sensory neurons, neurotransmitters, tyrosine kinases, ontogeny.
Studies over many years have established that sensory ganglia constitute a heterogeneous population of neurons [3, 7, 9, 24, 48, 95]. Sensory neurons are heterogeneous morphologically and histochemically, as well as in terms of receptor type and immunohistochemical characteristics [1, 4–6, 16, 17, 23, 50] and target organs [37, 57]. Most large neurons with myelinated fibers of the Aδ group contain 200-kDal neurofilament protein (NF200). Other small afferent neurons in the intervertebral ganglia are able to bind Griffonia simplicifolia isolectin B4 (IB4) [21, 84, 115]. Small-cell subpopulations are believed to transmit pain information, while large nerve cells conduct proprioceptive and tactile spikes [39, 87]. Sensory neurons also differ in terms of neurotransmitter composition. These include substance P, neurokinins, calcitonin gene-related peptide (CGRP), cholecystokinin, somatostatin, glutamate, galanin, and vasoactive intestinal peptide (VIP) [14, 24, 43, 50, 61, 99]. Some of these neuropeptides (CGRP, substance P, neurokinin A) are released from the same sensory endings, providing a “local efferent function.” Afferent neurons are traditionally regarded as perceiving only the actions of stimuli and transmitting exci-
List of abbreviations: VIP . . . . . . . . . . vasoactive intestinal peptide IB4 . . . . . . . . . . isolectin B4 CGRP . . . . . . . . calcitonin gene-related peptide NT . . . . . . . . . . neurotrophin NF200 . . . . . . . . 200-kDal neurofilament protein RET . . . . . . . . . . receptor tyrosine kinase IVG . . . . . . . . . . intervertebral ganglion TG . . . . . . . . . . trigeminal ganglion TRK . . . . . . . . . . tyrosine kinase SGVN . . . . . . . . sensory ganglion of vagus nerve BDNF . . . . . . . . brain-derived neurotrophic factor GDNF . . . . . . . . glial cell-derived neurotrophic factor GFRα1–4 . . . . . . α receptor of the GDNF family NO . . . . . . . . . . nitric oxide NOS . . . . . . . . . . NO synthase 1 Yaroslavl
State Medical Academy, Ministry of Health of the Russian Federation, Yaroslavl, Russia; e-mail:
[email protected]. 2 Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg, Russia.
440 0097-0549/15/4504-0440 ©2015 Springer Science+Business Media New York
Neurochemical Characteristics of Sensory Neurons during Ontogeny tation via efferent neurons or interneurons. However, more recent studies have demonstrated the existence of a special group of neurons: afferent neurons with efferent functions. Thus, some afferent fibers have purely afferent functions, while others have only local effector functions; finally, a third group of fibers can carry out both functions – both afferent and local effector [3, 30, 53]. 200-kDal Neurofilament and Isolectin B4 in Sensory Neurons Neurofilaments are the main structural component of the cell carcass, are expressed in neuronal cells, and are required for the function of axon transport [27, 103]. Neurofilaments contain three neuron-specific proteins with different molecular weights: light (68 kDal), intermediate (160 kDal), and heavy (200 kDal). NF200 is a marker for large neurons in intervertebral ganglia (IVG) with myelinated axons, which mount responses to mechanical stimuli [6, 93]. Thus, NF200 is present in 45% of neurons in the lower thoracic and upper lumbar ganglia in sheep, 15% of neurons in the lower lumbar ganglia, and 30% of neurons in the upper thoracic and lumbar ganglia in rats [5, 7, 99]. IB4 binds to cell surfaces via carbohydrates and is detected in sensory nerve cell populations whose fibers lack myelin [104, 115]. Small P2X3-immunoreactive neurons in sensory ganglia are able to bind IB4. IB4 binding is detected in the plasmalemma and diffusely in the cytoplasm [95]. Species differences have been found in relation to the numbers of neurons containing IB4 and NF200. In the lumbar ganglia in rats, some 40% of neurons bind IB4 and a similar proportion are immunoreactive to NF200 [19]. At the same time, 79% of cells in ganglia in sheep were IB4positive and 45% were NF200-positive. In contrast to rodents, IB4-positive nerve cells in sheep were colocated with NF200 [99]. There were also differences in the ratios of IB4- and BF200-immunoreactive neurons innervating different target organs. Thus, for example, in sensory ganglia in rats, IB4 was present in 20% and NF200 in 40% of neurons innervating the tibia. However, IB4 was present in a greater proportion of neurocytes innervating the skin (34%), NF200 being present in much the same proportion (43%). IB4binding was seen with small neurons and NF200 mainly to intermediate-sized cells [57]. Sensory Ganglion Neurons Containing Different Types of Calcium-Binding Proteins Calcium-binding proteins whose structures contain from two to six calcium-binding centers have now been described [2]. The most widely distributed in the nervous system are 28-kDal calbindin, calretinin, and parvalbumin, which are members of the EF family of calcium-binding proteins [13, 100]. Depending on the Ca2+ concentration, calcium-binding proteins undergo different interactions with their target proteins to regulate their activity [49, 101]. Calretinin plays an important role in regulating the level of cell excitability and synaptic transmission process-
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es, particularly the induction of long-term potentiation in hippocampal neurons [26]. Mutant mice lacking calretinin showed changes in the functional characteristics of neurons in the central nervous system, particularly an increase in the spike frequency of Purkinje cells, with shorter interspike intervals [32]. Calbindin functions not only as a calcium buffer, but also as a calcium sensor. On binding, calcium alters the conformation of protein molecules and activate myoinositol monophosphatase 1, the key enzyme in the inositol triphosphate pathway, and Ran-binding protein M [78, 100]. Calbindin, which binds calcium, can regulate intracellular responses to physiological stimuli and protect cells from calcium-mediated neurotoxicity [70]. Calbindin also plays an important role in protecting cells from apoptosis by inhibiting the key enzyme caspase 3 [18]. Among the factors controlling the development of synapses and their plasticity, maintenance of a defined concentration of Ca2+ ions s important, and calbindin plays a major role in this process [101]. In ganglia in adult rats, the proportions of calretinin- and calbindin-immunopositive neurons are 5–8% and 20–30%, respectively [12, 28]. Similar proportions of calbindinimmunopositive neurons are also seen in other sensory ganglia – the sensory ganglion of the vagus nerve (SGVN) and the trigeminal ganglion (TG) [98]. Sensory Neurons Containing Different Types of Tyrosine Kinase The family of tyrosine-specific protein kinases are catalytic receptor proteins crossing the membrane once. The catalytic domain is on the internal side of the plasma membrane. Ligand binding causes activation, transferring a phosphate group from ATP to the hydroxyl group of a tyrosine reside in particular proteins. Different types of tyrosine kinase are identified: A (TrkA), B (TrkB), and C (TrkC). TrkA and TrkC are highly sensitive to nerve growth factor (NGF) and neurotrophin 3 (NT3), while TrkB is sensitive to brain-derived neurotrophic factor (BDNF) [40]. NGF promotes the survival of sympathetic and sensory neurons derived from the neural crest [111]. In contrast to sympathetic cells, the survival of sensory ganglion nerve cells loses its dependency on NGF during the first few days of life. However, during the first two weeks of postnatal development, specific nociceptive neurons with non-myelinated and fine myelinated fibers are dependent on NGF [72]. In adult rats, about 40% of mature nerve cells in ganglia express the NGF-TrkA receptor, which prolongs the influence of NGF on mature sensory neurons [16, 83]. In adult individuals, NGF regulates the expression of substance P and CGRP in neurons in sensory ganglia, these often being colocated with TrkA [12, 114]. It has been suggested that NGF has direct or indirect influences on the ability of signals from target organs to reach neurons at embryonic developmental stages [90]. In addition, administration of NGF induces thermal and mechanical hyperalgesia, while decreases in NGF using blocking antibodies lead
442 to decreased sensitivity to pain stimuli [72]. Thus, in addition to supporting neuron survival during embryogenesis and determining specificity in the early postnatal ontogeny, NGF in adults regulates the functional properties of nociceptive sensory cells. NGF has been shown to play an important role in the survival of neurocytes at stages E11.5 and E15.5. Lack of NGF and TrkA during the embryonic period in mice increases the apoptosis of nerve cells in ganglia [117] and decreases non-myelinated fibers by more than 95% and myelinated nerve fibers, including type Aδ axons, by 50% [105]. Studies on mutant mice have shown that NGF is required not only for the survival of small and intermediate neurons, including peptidergic and non-peptidergic nociceptors, but also for the persistence of large TrkB- and TrkC-positive cells in sensory ganglia [90, 107]. BDNF and NT3 have been shown to affect the survival of sensory neurons during the embryonic period and the first two weeks of life [8, 33, 62]. Published data indicate that neonatal mice lacking BDNF show decreases in the numbers of TrkB- and TrkC-immunoreactive cells [74]. BDNF is also involved in movement coordination and balance processes [38, 58], in specifying slowly adapting mechanoreceptors [29], and in regulating excitability on transmission of pain stimuli [75, 88]. At the early stages of embryonic development, neurotrophin 3 is required for the survival of proprioceptive neurons in ganglia and the establishment of their connections with targets [67, 91]. During embryogenesis (E18.5), NT3 mutant mice were found to show decreases in the numbers of neurons containing substance P and CGRP, which was probably associated with the lack of any effect on the precursors of differentiating neurocytes [10]. During the postnatal period, NT3 is involved in the processes establishing synaptic transmission between primary afferents and motoneurons in the anterior horns on the spinal cord [31]. It has been suggested that NT3, acting via the TrkC receptor, has an analgesic action and is involved in modulating neuropathic pain [109]. In adult humans, TrkA is presented in 46%, TrkB in 29%, and TrkC in 24% of neurons in sensory ganglia. It has also been shown that TrkB is expressed in neurocytes of different sizes, TrkA mainly in small cells, TrkC in large cells [59]. In adult rodents, about 40% of neurons also contain TrkA. A large proportion of TrkA-positive neurons in rats are small cells, 92% of which contain CGRP. TrkBimmunoreactive neurons account for 26% in rat lumbar ganglia and 10% in thoracic ganglia. TrkC-containing neurons account for 10% of cells in the lumbar ganglia in rats [81, 83]. Electrophysiological studies have shown that TrkA is expressed mainly in nociceptors [41]. In adult rats, 98% of SGVN neurocytes express TrkB; TrkA and TrkC receptors are seen only on occasional neurons [60]. At the same time, immunoreactivity to different types of tyrosine kinase also depends on the target organ. Most sensory neurons innervating the skin contain TrkA (80%), while TrkC is present in a majority of cells addressed to muscles. A
Maslyukov, Porseva, Korzina, and Nozdrachev large proportion of neurons projecting to the body organs of contain TrkA and TrkB, which are colocated [40]. Despite the limited expression of TrkA in small neurons, in adult mice lacking the gene responsible for synthesizing TrkA show decreases in the numbers of all small-diameter neurocytes [105]. Two explanations for this have been suggested. The essence of the first is that all small neurons express TrkA, while IB4-containing neurons later stop expressing it [82]. The second is that IB4-binding neurons do not express it at all, their viability depending on TrkApositive nerve cells which produce neurotrophins by a paracrine mechanism. This latter is supported by data showing that BDNF and NT3 are released by many neurons in ganglia during development [40]. RET receptor tyrosine kinase is the signal component of receptors of the glial-derived neurotrophic factor (GDNF) family. Apart from this zone, there is also a ligand-binding region, the so-called α receptor of the GDNF family (GFRα1–4), located on the cell surface [11]. The RET-type receptor tyrosine kinase, which is detected in all non-peptidergic IB4-binding neurons with non-myelinated groups C fibers, accounts for up to 60% in rat sensory ganglia [22, 23]. About 50% of neurons in the lumbar ganglia of rats express GFRα1, 20% express GFRα2, and 20% express GFRα3 [60]. In adult mouse lumbar ganglia, more than 90% of small IB4positive neurons contain the RET receptor [46]. Published data indicate that more than 90% of RET-positive neurons in mice are of small and intermediate size [77]. RET receptors in the sensory ganglia of adult humans are seen in 79% of neurons, GFRα1 in 20%, GFRα2 in 51%, and GFRα3 in 32%. GFRα2 and GFRα3 coreceptors are typical of small cells, and GFRα 1 and RET of large and small cells [59]. RET and GFRα1 are coexpressed in 30–40% of adult rat SGVN neurons [60]. RET immunoreactivity is detected in 75% of intermediate and small neurocytes in the human TG; 65% contain GFRα1, colocated with RET receptors in half. Both RET- and GFRα1-positive neurons in the TG have been shown to coexpress CGRP [97]. Neurotransmitters of Sensory Neurons Substance P and CGRP. About 60% of all IVG neurons are small-diameter cells with non-myelinated C-type fibers and myelinated Aδ-type fibers [80]. Electrophysiological studies have established that about 90% of these cells in mice, rats, monkeys, and humans have nociceptive functions. Small cells are divided onto two classes on the basis of neurochemical phenotype [106]: peptidergic and non-peptidergic. About half of them express CGRP or substance P, as well as p75 neurotrophic receptor and TrkA, and NGF-specific receptor tyrosine kinase. The central processes of these neurons extend to the superficial plate of the posterior horn of the spinal cord, where many afferent terminals are located. TrkA is observed in about 80% of substance P-containing neurons in the lumbar ganglia of rats [61]. There are differences in neurochemical composition between neurons in the IVG and SGVN. The vast majority of
Neurochemical Characteristics of Sensory Neurons during Ontogeny SGVN neurons contain substance P. The proportion of substance P-immunoreactive neurons in the rat IVG is no more than 10% [6, 50], though it can reach 42% in sheep [99]. CGRP is present in both the central and peripheral nervous system. CGRP-containing neurons are members of the class of cells with C-, Aδ, and Aα/β fibers [80]. Two forms of CGRP are known: alpha-CGRP is predominantly present in Aδ and C fibers of the trigeminal nerve and IVG neurons; the beta isoform of this peptide is located mainly in in intestinal nerve plexuses [35]. Histochemical studies of cerebral perivascular nerve endings in TG cells showed that more than half of neurons contain CGRP [36, 108]. CGRP is the most widespread neuropeptide found in the primary afferent nerve cell subpopulation, accounting for 40–50% of ganglion cells, most of which are nociceptors [69]. The number of CGRP-positive cells varies strongly depending on target organ [21], spinal cord segment [89], and even between members of a single species [112]. CGRP is detected in 53% of neurons innervating the skin and in 23% of sensory neurons sending axons to bones. CGRP is detected primarily in neurons of small and intermediate size [57]. In the vagus ganglia of mammals, a greater proportion of CGRP-immunoreactive neurons is present in the cranial sensory ganglion as compared with the caudal ganglion. In cats, the CGRP-immunoreactive neurons in these ganglia amount to 72% and 42%, compared with 30% and 10% in rats [43, 56, 113]. CGRP is a powerful vasodilator [76]. Depending on dose, it suppresses neurogenic smooth muscle contraction [45] or, contrarily, contracts them [47]. In addition, CGRP, along with substance P, initiates the development of neurogenic gastric inflammation and hyperalgesia [65], increases vascular and tissue permeability, and activates connective tissue cells in peripheral tissues [52]. At the same time, CGRP can be an inhibitor of substance P. Thus, the coexistence of CGRP with substance P can be explained by the increase in substance P-mediated transmission, due to its degradation [71]. It has been suggested that substance P and CGRP in lumbar ganglion neurocytes are involved in regulating intraganglion blood flow [64]. In the SGVN, about half of CGRP-immunoreactive neurons contain substance P and, in turn, almost all substance P-immunoreactive neurons are CGRP-immunoreactive [51, 55]. The adult human TG contains about 40% CGRP-immunoreactive neurocytes; 18% of nerve cells show substance P immunoreactivity. Less than 5% of CGRPimmunopositive neurons in the TG are NOS-positive [108]. In adult rat spinal cord ganglia, 84% of CGRP-immunoreactive cells are colocated with TrkA and 6% with TrkC; coexpression with TrkB in this cell type was not seen [61]. Nitric Oxide Sensory neuron neurotransmitters include small molecules, particularly nitric oxide (NO). Quite contradictory data have been obtained demonstrating the proprioceptive and antinociceptive actions of NO in sensory neurons.
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NO can play a role in sensitization processes due to cGMP synthesis and subsequent phosphorylation of specific membrane proteins mediated by protein kinase C. NO can also activate different types of TRP channels, including TRPC5, TRPV1, TRPV3, TRPV4, and TRPA1. Activation of the enzyme synthesizing nitric oxide – NO synthase (NOS) – can potentiate tetrodotoxin-resistant Na+ currents, facilitating inflammatory hyperalgesia and decreasing pain thresholds. The possible antinociceptive action of NO is associated with the opening of ATP-sensitive K+ channels on nociceptor membranes, leading to hyperpolarization and decreases in excitability [94]. NOS is linked with NADPH diaphorase. In rats, these cells account or less than 2% of all neurons in the sensory ganglia from C1 to T4 and from L2 to the last sacral vertebra, though they are numerous (up to 50%) from T5 to L1 [110]. In sheep, the proportion of NOS-containing neurons in ganglia from T13 to L2 is 44% [99]. The adult rat TG contains a small proportion of NOSpositive neurons, while the SGVN has a significantly greater proportion – up to 30% [116]. In humans, the TG contains 15% NOS-positive neurons. Also in humans, most vagus nerve sensory neurons are NO-positive and substance P-negative [108]. Age-Related Changes in Sensory Neurons with Different Neurochemical Characteristics Neurons containing NF200, IB4, and different types of calcium-binding proteins during ontogeny. IB4 is not detected in IVG neurons in rodent embryos and the first IB4-positive neurons appear only in neonatal animals. During the first week of life, the proportions of IB4-positive cells increases rapidly, such that the proportion of these cells by the end of the second week life reaches the adult level [98]. However, some data indicate that IB4-containing neurons are also present during the embryonic period, at least on day 17 of embryonic development (stage E17) [19]. NF200-immunopositive neurons are detected during the embryonic period, though the proportion of NF200-containing neurons in different sensory ganglia change in different directions. The proportions of these cells in the IVG and SGVN between embryonic day 16 and the moment of birth increases from 12% to 30% and from 3% to 12%, respectively, though it decreases from 49% to 31% in the TG. Nonetheless, the proportion of NF200 neurons in the IVG and TG increases by one third during the first two weeks of life, with a 2.5-fold increase in the SGVN [98]. Age-related changes in the quantitative content of NF200 neurons in the IVG depend on the segmental level up to day 20 of life, after which the subpopulation of immunoreactive neurons becomes relatively stable and remains so to age one year [5]. Cell cultures from chick embryo sensory ganglia established at stage E6 of embryonic development have been shown not to contain calretinin immunoreactivity, while at stage E10 there was strong immunopositivity
444 immediately after attachment of cultures to Petri dishes. The authors suggested that in vivo calretinin expression was similar to in vivo expression and that the expression of the gene responsible for calretinin synthesis depends on the establishment of connections with target organs [63]. Studies using immunohistochemical methods have shown that calretinin is expressed in the rat IVG during two phases of development – between stages E11 and E14, when calretinin is present in about 75% of cells, with a second phase, from day 17 of embryonic development and throughout the whole of postnatal ontogeny, when calretinin is present in fewer than 8% of nerve cells. Between these periods, no calretinin was detected in neurocytes in ganglia. It was suggested that calretinin at the early stages of development acts as a regulator of Ca2+ until the point at which neurotrophins start to play a role in the finer regulation of the concentration of this ion [12]. Characteristics of the Developmental Expression of Different Types of Tyrosine Kinase TrkA starts to be expressed in IVG neurons at stage E12. A small proportion of RET-positive, TrkA-negative neurons are seen at stage E12. However, a large proportion of RET-positive nerve cells arise from TrkA-positive neurons. At stage E16, many small and intermediate TrkAimmunopositive neurons start to express RET [82]. After birth (between day 5 and day 10 of life), TrkA expression in these cells starts to decrease. Final separation between TrkA and RET populations is completed between week 2 and week 3 of life [77, 82]. In adult mice, TrkA and RET coexpression is seen in about 9% of IVG neurons [46]. NGF controls the expression of RET and its coreceptors GFRα1 and GFRα2. Between the second and third weeks of life, NGF acts via RET to suppress TrkA expression in RET/TrkA-positive IVG neurons, thus increasing the population of RET-containing neurons. RET in the IVG at the postnatal stages regulates the maturation of non-peptidergic nociceptors and is also required for their survival, for trophics, and for maintaining normal soma size [46, 77]. In RET deficiency, this type of neuron undergoes hypertrophy and peptide-containing nociceptors and proprioceptive neurons do not change. In the mouse epidermis, lack of RET leads to decreases in the numbers of peripheral processes of nonpeptidergic neurons. In addition, the proportion of TrkAimmunoreactive, IB4-expressing neurons in the IVG in RET mutant mice at the P14 stage increases more than sixfold, while the proportion of neurocytes coexpressing TrkA and CGRP decreases four-fold [77]. RET receptors have been shown to take part in neurogenic inflammation and to increase animals’ sensitivity to cold and mechanical stimuli, but not to affect sensitivity to heat [44]. The expression of different types of Trk receptor in rodent IVG neurons is first seen during the neurogenesis period (E10–E13), which indicates that cells are already differentiated due to activation of certain genes [42]. Studies using immunohistochemical methods have shown
Maslyukov, Porseva, Korzina, and Nozdrachev that some TrkA-positive neurons in the IVG of the cervical and thoracic segments in mouse embryos are first detected at stage E10.5 [96, 117]. At stage E11, about 20% of IVG neurocytes show TrkA immunoreactivity, and the number of these cells increases sharply during the next two days (to 80%), with no subsequent change to birth [42]. During postnatal ontogeny, the proportion of TrkA-containing neurons decreases. By day 14–21 of life, the number of these neurocytes decreases almost two-fold and then remains unchanged [20, 82]. In the mouse TG at stage E10.5, only occasional TrkA-con cells are seen; by E12.5, the proportion of these cells increases to 75%. By stage E15.5, the number of TrkA-positive neurons decreases to 50% [54]. During embryogenesis in rats, the number of TrkA-containing neurocytes in the TG increases during the period from E12 to E18 [15]. A sharp decrease in the proportion of TrkA-expressing neurons was seen after the second day of life, when IVG neurocytes lose their dependence on NGF for survival. This fact indicates that the population of TrkA-immunoreactive nerve cells decreases as a result of a reduction in in NGF receptor synthesis rather than from cell death [82]. Published data provide evidence that sensory neurons in the peripheral nervous system terminate TrkA expression in the time period between neurogenesis and the establishment of connections with target organs [117, 118]. This probably allows local sources of neurotrophins to maintain neurocytes until they gain access to target organ trophic factors. TrkA expression is also seen in a proportion of neurons in adult individuals, i.e., neurotrophins continue to act on mature cells [119]. Some TrkA-immunoreactive neurons differentiate into IB4-containing neurons. Immunohistochemical studies first demonstrated IB4 in the lower lumbar ganglia in mice in small neurons in neonatal animals, colocated with TrkA in most cells [82]. About 40% of the neurons in the thoracic and lumbar ganglia show TrkB immunoreactivity in stage E11 mice. At stages E13–E15, 8–10% of rat and mouse IVG neurocytes are TrkB-immunopositive [73]. In mice, the proportion of such nerve cells at birth was the same [42], while in adult rats the proportion of TrkB-positive cells increased in the lumbar ganglia bur remained unaltered in the thoracic ganglia [81, 83]. At stage E10.5, TrkB was detected in 15% of neurocytes in the mouse embryo TG, with an eight-fold increase by E11.5 and a decrease to 40% by E15.5 [54]. In mice at stages E10.5–E11, about 70% of neurons in the thoracic and lumbar ganglia and virtually all nerve cells in the cervical ganglia express TrkC [42, 96]. In the lumbar and sacral ganglia at stage E13, proportion of such neurons decreased almost seven-fold and subsequently remained without change [42]. In TG neurons in the same animals at stage E10.5, TrkC was expressed in about 35% of cells, with a five-fold increase by E11.5 and a subsequent decrease to the undetectable by E15.5 [54]. The peak of TrkB and TrkC expression in rat TG neurons was seen at
Neurochemical Characteristics of Sensory Neurons during Ontogeny E12, while only occasional cells were seen at E16–E18 [15]. Other studies demonstrated that the expression of TrkC in the TG and the thoracic and lumbar IVG in mice, like that of TrkB, is seen in postmitotic nerve cells [42, 54]. It has been suggested that the maximal levels of TrkB and TrkC expression in TG neurons at stages E11–E12 can be explained on the basis of the higher dependence of neuron survival on BDNF and NT3, which at this time are secreted in large quantities. At the same time, the relatively small percentage of TrkA-positive neurocytes at these same stages is associated with the low sensitivity to these neurotrophins. At stage E12.5, when the level of TrkA expression in neurocytes peaks, as well as at later stages of embryonic development, nerve cell survival starts to be maintained mainly by NGF [34, 54]. Some authors have also suggested that a decrease in the number of neurons containing TrkA, TrkB, and TrkC during neurogenesis is related to the apoptosis of TG cells, probably due to competition for neurotrophins [42, 54]. At stage E11.5, 75% of neurocytes in the lumbar IVG in mice show TrkB and TrkC coexpression [66]. The proportion of such neurons decreases sharply to 10% at stage E12.5, while colocalization is not seen at stage E14.5. In the TG of mouse embryos at E11.5, the proportion of TrkA/TrkCexpressing nerve cells is 9%, that of TrkB/TrkB-expressing nerve cells is 10%, and that Of TrkB/TrkA-expressing nerve cells is 24%; at E12.5, the proportions are 5%, 6%, and 2% respectively. At later stages of embryo development, neurons containing more than one type of Trk receptor are not seen [54]. Coexpression of different types of tyrosine kinase in rat embryo TG cell cultures is seen at stage E16 [86]. It has been suggested that the disappearance of neurocytes expressing multiple forms of Trk receptors is associated with switching to the expression of any one type after neurogenesis [92]. Developmental Changes in the Neurotransmitter Composition of Sensory Ganglion Neurons There are very few published data on developmental changes in the neuropeptide composition of sensory neurons. In rat IVG neurons, substance P is first detected at stage E19.5 and the levels seen in adult animals are rarely reached by birth [48, 79]. The proportion of neurons with CGRP immunoreactivity in the cervical IVG increases during the period from the middle to the late fetal phases [85]. At stage E15, CGRP is visualized in occasional cells in the rat IVG, and there are very few such cells at stage E18.5 [48] or at stage E15 in mice [82]. By the first day of life, the proportion of these cells is established at the level of 20%. Frfom this day, there is a high level of TrkA/CGRP colocalization in nerve cells [82]. During the first week of life, the proportion of CGRP-containing neurons increases to 30% and then remains unaltered [6, 48]. It is interesting that neurochemical phenotypes in relation to substance P and CGRP are established during the embryonic period before formation of contacts with the target organs of innervation.
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Cultivation of embryonic IVG neurons in the absence of target organs has no effect on the expression of the above neuropeptides in these cells [48]. However, data on developmental changes in CGRP in peripheral fibers are contradictory. CGRP-containing afferent fibers are present in the airways from the moment of birth; the content of this neuropeptide in rats is not constant, but decreases from the moment of birth to day 21 of life [25]. At the same time, the CGRP content in the rat atrium increases after birth, becomes maximal by day 60, and then decreases [68]. Conclusions Sensory ganglion neurons constitute a heterogeneous population differing in terms of morphological, receptor, and immunohistochemical characteristics. During ontogeny, different populations of afferent ganglia develop at different times. The growth and development of sensory neurons, especially during the embryonic period, occur under the influence of neurotrophins, particularly nerve growth factor, brain- and glial cell-derived growth factor, and neurotrophin NT3. The existence of heterogeneous populations of afferent neurons differing in terms of receptor and neurochemical composition allows selective influences to be exerted on individual groups of nerve cells using pharmacological agents, which is important, for example in the treatment of chronic pain syndromes. Studies of the development of afferent neurons will not only provide a better understanding of the principles of the development of neural networks, but will also increase our knowledge of nociceptive mechanisms. This work was supported by the Russian Foundation for Basic Research (Grant Nos. 12-04-00621, 13-04-00059) and the Federal Targeted Program on Scientific and Teaching Staff for an Innovative Russia in 2009–2013 (Agreements 8290, 8566, and 8603). REFERENCES 1. 2. 3. 4.
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