Role of neurotrophin signalling in the differentiation of neurons from dorsal root ganglia and sympathetic ganglia

Manipulation of neurotrophin (NT) signalling by administration or depletion of NTs, by transgenic overexpression or by deletion of genes coding for NTs and their receptors has demonstrated the importance of NT signalling for the survival and differentiation of neurons in sympathetic and dorsal root ganglia (DRG). Combination with mutation of the proapoptotic Bax gene allows the separation of survival and differentiation effects. These studies together with cell culture analysis suggest that NT signalling directly regulates the differentiation of neuron subpopulations and their integration into neural networks. The high-affinity NT receptors trkA, trkB and trkC are restricted to subpopulations of mature neurons, whereas their expression at early developmental stages largely overlaps. trkC is expressed throughout sympathetic ganglia and DRG early after ganglion formation but becomes restricted to small neuron subpopulations during embryogenesis when trkA is turned on. The temporal relationship between trkA and trkC expression is conserved between sympathetic ganglia and DRG. In DRG, NGF signalling is required not only for survival, but also for the differentiation of nociceptors. Expression of neuropeptides calcitonin gene-related peptide and substance P, which specify peptidergic nociceptors, depends on nerve growth factor (NGF) signalling. ret expression indicative of non-peptidergic nociceptors is also promoted by the NGF-signalling pathway. Regulation of TRP channels by NGF signalling might specify the temperature sensitivity of afferent neurons embryonically. The manipulation of NGF levels “tunes” heat sensitivity in nociceptors at postnatal and adult stages. Brain-derived neurotrophic factor signalling is required for subpopulations of DRG neurons that are not fully characterized; it affects mechanical sensitivity in slowly adapting, low-threshold mechanoreceptors and might involve the regulation of DEG/ENaC ion channels. NT3 signalling is required for the generation and survival of various DRG neuron classes, in particular proprioceptors. Its importance for peripheral projections and central connectivity of proprioceptors demonstrates the significance of NT signalling for integrating responsive neurons in neural networks. The molecular targets of NT3 signalling in proprioceptor differentiation remain to be characterized. In sympathetic ganglia, NGF signalling regulates dendritic development and axonal projections. Its role in the specification of other neuronal properties is less well analysed. In vitro analysis suggests the involvement of NT signalling in the choice between the noradrenergic and cholinergic transmitter phenotype, in the expression of various classes of ion channels and for target connectivity. In vivo analysis is required to show the degree to which NT signalling regulates these sympathetic neuron properties in developing embryos and postnatally. U.E. is supported by the DFG (Er145-4) and the Gemeinnützige Hertie-Stiftung.     

Functional roles of neurotrophin 3 in the developing and mature sympathetic nervous system

Nerve growth factor (NGF) is a potent regulator of sympathetic neuronal function in both developing and adult animals. This article reviews the evidence published in recent years indicating that another member of the NGF family, neurotrophin 3 (NT3), plays both a complementary and overlapping role in the development and maturation of sympathetic neurons. In migratory neural crest cells, expression of the high-affinity receptor, trkC, and promotion of mitosis by NT3 suggest an involvement in gangliogenesis, since sympathetic neuroblasts express both NT3 and trkC and require NT3 for their proliferation, differentiation, and survival, it has been proposed that the factor acts at this developmental stage as an autocrine or paracrine factor. However, NT3 also acts in parallel with NGF to promote the survival of postmitotic neurons during late development. Both trkC and trkA are expressed in sympathetic neurons and function as high-affinity receptors for NT3. NT3 is synthesized in sympathetic effector tissues and the endogenous factor is retrogradely transported to accumulate within the cell soma. Thus, in addition to its role in the differentiation of sympathetic neurons, NT3, like NGF, is also an effector tissue-derived neurotrophic factor for these neurons in maturity.     

Neurotrophin-3 promotes the survival of a limited subpopulation of cutaneous sensory neurons

In the chick embryo, exogenous neurotrophin-3 (NT3) is sufficient to promote the differentiation of proprioceptive afferents even in the absence of limb muscle targets. To determine if NT3 can promote the differentiation of this phenotype in afferents with cutaneous targets, we analyzed the effects of chronic NT3 on cutaneous and muscle sensory neurons that express trkC, a receptor for NT3. In normal embryos, retrograde labeling and immunohistochemistry showed that about 75% of large-diameter muscle afferents express trkC, whereas only about 7% of large-diameter cutaneous afferents express this protein. After chronic treatment with NT3 during the cell death period, both populations of trkC(+) neurons were increased approximately twofold. Because this treatment is known to block cell death in sensory neurons, these results indicate that NT3 can promote the survival of both proprioceptive afferents and cutaneous afferents. To examine the phenotype of the cutaneous afferents rescued by NT3, we analyzed their projections and connections using transganglionic labeling and electrophysiological recording. The results indicate that exogenous NT3 neither altered the pattern of spinal projections nor caused cutaneous afferents to form monosynaptic connections with motor neurons. These results demonstrate that selective cell death does not contribute to the modality-specific pattern of spinal innervation and suggest that proprioceptive afferents may innervate muscle selectively.     

Primary afferent neurons intrinsic to the guinea-pig intestine,like primary afferent neurons of spinal and cranial sensory ganglia,bind the lectin,IB4

The plant lectin, IB4, binds to the surfaces of primary afferent neurons of the dorsal root and trigeminal ganglia and is documented to be selective for nociceptive neurons. Physiological data suggest that the intrinsic primary afferent neurons within the intestine are also nociceptors. In this study, we have compared IB4 binding to each of these neuron types in the guinea-pig. The only neurons in the intestine to be readily revealed by IB4 binding have Dogiel-type-II morphology; these neurons have been previously identified as intrinsic primary afferent neurons. Most of the neurons that are IB4-positive in the myenteric plexus are calbindin-immunoreactive, whereas those in the submucosal ganglia are immunoreactive for NeuN. The neurons that bind IB4 strongly have a similar appearance in enteric, dorsal root and trigeminal ganglia. Binding is to the cell surface, to the first part of axons and to cytoplasmic organelles. A low level of binding was found in the extracellular matrix. A few other neurons in all ganglia exhibit faint staining with IB4. Strongly reactive neurons are absent from the gastric corpus. Thus, IB4 binding reveals primary afferent neurons with similar morphologies, patterns of binding and physiological roles in enteric, dorsal root and trigeminal ganglia.This work was supported by a grant from the National Health and Medical Council of Australia.     

Programmed cell death in zebrafish rohon beard neurons is influenced by TrkC1/NT-3 signaling

Rohon Beard (RB) cells are embryonic primary sensory neurons that are removed by programmed cell death during larval development in zebrafish. RB somatosensory functions are taken over by neurons of the dorsal root ganglia (DRG), suggesting that RB cell death may be triggered by the differentiation of these ganglia, as has been proposed to be the case in Xenopus. However, here we show that the timing of RB cell death correlates with reduced expression of trkC1, the receptor for neurotrophin NT-3, but not with the appearance of DRG, which differentiate only after most RB cells die. trkC1 is expressed in subpopulations of RB neurons during development, and cell death is initiated only in trkC1-negative neurons, suggesting a role for TrkC1 and its ligand, NT-3, in RB cell survival. In support of this, antibodies that deplete NT-3 induce RB cell death while exogenous application of NT-3 reduces death. In addition, we show that RB cell death can be prevented using a caspase inhibitor, zVADfmk, showing that during normal development, RB cells die by a caspase-dependent programmed cell death pathway possibly triggered by reduced signaling via TrkC1.     

Binding of Isolectin IB4 to Neurons of the Mouse Enteric Nervous System

The plant lectin, IB4, binds to primary afferent neurons of dorsal root and trigeminal ganglia, where it is selective for nociceptive neurons. In the enteric nervous system of the guinea-pig IB4 labels intrinsic primary afferent neurons, which are believed to have roles as nociceptors. Here we investigate whether IB4 binding is also a marker of intrinsic primary afferent neurons in the mouse. Neurons that bound IB4 were common in the enteric plexuses of the small intestine and colon. Labeled neurons were rare in the stomach, and absent from the esophagus and gallbladder. Binding was to the cell surface, initial parts of axons and to clumps in the cytoplasm. Similar binding occurred on small and medium sized neurons of dorsal root, nodose and trigeminal ganglia. In the enteric nervous system, IB4 revealed large round or oval (type II) neurons, type I neurons with prominent laminar dendrites and small neurons of myenteric ganglia. The type II neurons were immunoreactive for calretinin, and some type I neurons were immunoreactive for nitric oxide synthase. Most neurons in the submucosal ganglia bound IB4, and some of these were vasoactive intestinal peptide immunoreactive. Thus IB4 binds to specific subgroups of enteric neurons in the mouse. These include intrinsic primary afferent neurons, but other neurons, including secretomotor neurons, are labeled. The results suggest that IB4 is not a specific label for enteric nociceptive neurons.     

Distribution and changes with age of calcitonin gene-related peptide- and substance P-immunoreactive nerves of the rat urinary bladder and lumbosacral sensory neurons

In the distal parts of the urinary tract, nerves containing calcitonin gene-related peptide (CGRP) or substance P (SP) are sensory with their cell bodies located in lumbosacral dorsal root ganglia. These two neuropeptides are recognised as being present in pelvic sensory nerves, and may be involved in the mediation of pain, stretch and/or vasodilatation. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive (-ir) for CGRP and SP in the urinary bladder and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made for CGRP-ir and SP-ir fibres innervating the dome, body and base of the urinary bladder. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for CGRP and SP. There were very few immunoreactive axons in the dome and the overall density of innervation increased progressively towards the base of the bladder. The density of innervation in the aged rats revealed a slight reduction in CGRP and SP innervation of the detrusor muscle but was otherwise comparable to the young group. However, immunostaining of the lumbosacral dorsal root ganglia revealed that the percentage of CGRP- and SP-ir neuronal profiles showed a significant (P < 0.05) reduction from (mean +/- S.D) 44.5 +/- 2; 23.3 +/- 2 in young adult to 25.0 +/- 2.9; 14.8 +/- 1.6 in aged rats, respectively. These findings suggest that the involvement of CGRP and SP in urinary bladder innervation is relatively unchanged in old age, but their expression in dorsal root ganglion neurons is affected by age. The afferent micturition pathway from the pelvic region via these lumbosacral ganglia may be perturbed as a result.     

P2X_3受体在感觉神经节的表达

用免疫组织化学与原位杂交研究 P2 X3受体在背根神经节、三叉神经节和结状神经节的分布。结果显示 :1.原位杂交 :在三种感觉神经节中 ,95 %左右的神经节细胞为 P2 X3m RNA阳性 ,中、小型神经节细胞的杂交信号一般要比大型的神经节细胞强一些。 2 .免疫组织化学 :免疫组织化学结果与原位杂交结果基本一致。此外 ,在各神经节内 ,均显示出许多P2 X3免疫阳性神经纤维 ,在足掌表皮也显示许多 P2 X3免疫反应阳性纤维。结果提示 :P2 X3不仅参与机体的痛觉的形成 ,还可能参与其它感觉 ,如本体感觉等的形成     

Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog

Summary In this study, immunohistochemistry for neuronal nitric oxide synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and nitric oxide synthase radioassay were used to study the occurrence, number and distribution pattern of nitric oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric oxide synthase immunolabelling was present in a large number of small- (area <1000 μm²) and medium-sized (area 1000–2000 μm²) as well as in a limited number of large-sized (area >2000 μm²) neurons. Although neuronal nitric oxide synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized nitric oxide synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of nitric oxide synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of nitric oxide synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central nitric oxide synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic nitric oxide synthase activity, determined by conversion of [³H]arginine to [³H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Heme oxygenase-2 in primary afferent neurons of the guinea-pig

Sensory ganglia (trigeminal, jugular, nodose, cervical and lumbar dorsal root ganglia) of the guineapig were investigated for the presence of a constitutive carbon monoxide-generating enzyme, heme oxygenase-2 (HO-2). A 36-kDa HO-2-immunoreactive protein was identified by Western blotting in protein extracts from dorsal root ganglia and localized by immunohistochemistry to all neuronal perikarya, including both substance P-positive and substance P-negative neurons, in all ganglia investigated. This ubiquitous distribution points to a general requirement for HO-2 in primary afferent neurons rather than to an association with a specific functionally defined subpopulation. Neither the axons of the sensory neurons nor their peripheral terminals in the skin and around visceral arteries were HO-2 immunoreactive. Explants of dorsal root ganglia with crushes placed on the dorsal roots showed accumulation of the neuropeptide, substance P, at the ganglionic side of the crush, but these axons were non-reactive to HO-2, indicating that there is no substantial transport of HO-2 towards the central ending of these sensory neurons. Collectively, the findings suggest that HO-2 exerts it major effects within the sensory ganglia themselves.     

Distribution and changes with age of nitric oxide synthase-immunoreactive nerves of the rat urinary bladder, ureter and in lumbosacral sensory neurons

In the distal parts of the urinary tract, nerves containing nitric oxide (NO) are either postganglionic parasympathetic nerves, with cell bodies in the major pelvic ganglia, or sensory nerves with cell bodies in the lumbosacral dorsal root ganglia. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive for neuronal nitric oxide synthase (NOS) in the urinary bladder, distal ureter and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made of NOS fibres innervating the dome, body and base of the urinary bladder and distal ureter. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for NOS. The dome and the body regions, in both age groups, contained no NOS-immunoreactive axons. The bladder base and distal ureter in young adults showed sparse to moderate numbers of fibres immunoreactive to NOS within the urothelium and in the subepithelium and muscle coat. In the aged rat there were slight reductions in the densities of NOS-immunoreactive nerves in all three regions. In the lumbosacral dorsal root ganglia, the percentage of NOS-immunoreactive neuronal profiles showed a significant reduction from 4.6 +/- 0.2% in young adult to 2.7 +/- 0.2% (means +/- S.E.M) in aged rats. These findings suggest that the effects of NO on the bladder and distal ureteric musculature and also its expression in dorsal root ganglion neurons are affected in aged rats and that the micturition reflex may be perturbed as a result.     

Light- and electron-microscopic study of synaptic connections in the paracervical ganglion of the female rat: special reference to calcitonin gene-related peptide-, galanin- and tachykinin (substance P and neurokinin A)-immunoreactive nerve fibers and terminals

Nerve fibers and varicosities in the pelvic paracervical ganglia (PG) are immunoreactive for the neuropeptides calcitonin gene-related peptide, galanin, and the tachykinins substance P and neurokinin A. Many of these fibers and varicosities are capsaicin-sensitive, originate in dorsal root ganglia and, thus, are considered to be primary afferent fibers. Numerous immunoreactive varicosities are pericellular to principal neurons in the PG. The present study examines the ultrastructure of calcitonin gene-related peptide-, galanin-, substance P-, and neurokinin A-immunoreactive nerve fibers and varicosities in the ganglia to determine their relationships to principal neurons and their synaptic connectivity. Paracervical ganglia of female rats were processed for light-microscopic immunohistochemistry using antisera against synapsin I, as a nerve terminal marker, and microtubule-associated protein-2 to define soma and dendrites. The rationale for performing this co-immunohistochemical analysis was to reveal the relationship between nerve endings and principal neurons. Synapsin I endings were predominantly axosomatic with fewer being axodendritic. Other ganglia were processed for electron-microscopic immunohistochemistry using both standard immunogold and peroxidase-anti-peroxidase procedures. Unmyelinated fibers and varicosities immunoreactive for calcitonin gene-related peptide, galanin, and the tachykinins were routinely observed in the interstitium between neuron somas. Numerous immunoreactive axon profiles were present in small groups that were ensheathed by Schwann cells. Immunoreactive fibers and varicosities were also observed within the satellite-cell sheath of the neuron soma and often intimately associated with the membrane of the soma, somal protrusions, or with the proximal part of a dendrite. Membrane specializations, indicative of synaptic contacts, between the fibers and the principal neurons were observed. It is suggested that these peptide-immunoreactive sensory fibers and varicosities are involved in regulation of activity in the PG.     

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Calretinin-immunoreactive nerves in the uterus, pelvic autonomic ganglia, lumbosacral dorsal root ganglia and lumbosacral spinal cord

Nerves containing the calcium-binding protein calretinin have been reported in several organs but not in female reproductive organs and associated ganglia. This study was undertaken to determine if nerves associated with the uterus contain calretinin and the source(s) of calretinin-synthesizing nerves in the rat (are they sensory, efferent, or both?). Calretinin-immunoreactive nerves were present in the uterine horns and cervix where they were associated with arteries, uterine smooth muscle, glands, and the epithelium. Calretinin-immunoreactive terminals were apposed to neurons in the paracervical ganglia; in addition, some postganglionic neurons in this ganglion were calretinin positive. Calretinin perikarya were present in the lumbosacral dorsal root ganglia, no-dose ganglia, and lumbosacral spinal cord. Retrograde axonal tracing, utilizing Fluorogold injected into the uterus or paracervical parasympathetic ganglia, revealed calretinin-positive/Fluorogold-labeled neurons in the dorsal root and nodose ganglia. Also, capsaicin treatment substantially reduced the calretinin-positive fibers in the uterus and pelvic ganglia, thus indicating the sensory nature of these fibers. The presence of calretinin immunoreactivity identifies a subset of nerves that are involved in innervation of the pelvic viscera and have origins from lumbosacral dorsal root ganglia and vagal nodose ganglia. Though the exact function of calretinin in these nerves is not currently known, calretinin is likely to play a role in calcium regulation and their function.     

Hot receptors in the brain

Background

The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.

Results

Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X₃. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.

Conclusion

The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.