Relative sparing of calcitonin gene-related peptide-containing primary sensory neurons following neonatal capsaicin treatment in the rat

Calcitonin gene-related peptide (CGRP)-containing sensory neurons projecting to viscera or skin were detected by immunocytochemistry combined with fluorescent tracer in the dorsal root ganglia (Th9-10) of rats 5-6 weeks old treated neonatally with capsaicin. The number of CGRP-like immunoreactive (IR) cells were reduced by 50-60% with capsaicin treatment. Visceral CGRP-IR sensory neurons were shown to be more sensitive than cutaneous ones, which was also supported by the fact that CGRP-IR fibers in the stomach were completely diminished while epidermal CGRP-IR fibers were spared.     

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ～25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.     

Dye-filling of the amphid sheath glia: implications for the functional relationship between sensory neurons and glia in Caenorhabditis elegans

The nervous system is composed of cells including neurons and glia. It has been believed that the former cells play central roles in various neural functions while the latter ones have only supportive functions for neurons. However, recent findings suggest that glial cells actively participate in neural activities, and the cooperation between neurons and glia is important for nervous system functions. In Caenorhabditis elegans, amphid sensory organs in the head also consist of sensory neurons and glia-like support cells (amphid socket and amphid sheath cells). Ciliary endings of some sensory neurons exposed to the environment detect various chemicals, molecules and signals, and the cilia of some neurons can also take up fluorescent dyes such as DiI. Here, we show that the amphid sheath glia are also stained with DiI and that its uptake by the amphid sheath cells correlates with DiI-filling of sensory neurons, suggesting that the amphid sheath glia might interact with sensory neurons. Furthermore, the localization of the amphid sheath cell reporter F52E1.2SP::YFP is abnormal in che-2 mutants, which have defective cilia. These findings imply that sensory neurons might affect amphid sheath glia functions in the amphid sensory organ of C. elegans.     

SCN2B in the Rat Trigeminal Ganglion and Trigeminal Sensory Nuclei

The beta-2 subunit of the mammalian brain voltage-gated sodium channel (SCN2B) was examined in the rat trigeminal ganglion (TG) and trigeminal sensory nuclei. In the TG, 42.6 % of sensory neurons were immunoreactive (IR) for SCN2B. These neurons had various cell body sizes. In facial skins and oral mucosae, corpuscular nerve endings contained SCN2B-immunoreactivity. SCN2B-IR nerve fibers formed nerve plexuses beneath taste buds in the tongue and incisive papilla. However, SCN2B-IR free nerve endings were rare in cutaneous and mucosal epithelia. Tooth pulps, muscle spindles and major salivary glands were also innervated by SCN2B-IR nerve fibers. A double immunofluorescence method revealed that about 40 % of SCN2B-IR neurons exhibited calcitonin gene-related peptide (CGRP)-immunoreactivity. However, distributions of SCN2B- and CGRP-IR nerve fibers were mostly different in facial, oral and cranial structures. By retrograde tracing method, 60.4 and 85.3 % of TG neurons innervating the facial skin and tooth pulp, respectively, showed SCN2B-immunoreactivity. CGRP-immunoreactivity was co-localized by about 40 % of SCN2B-IR cutaneous and tooth pulp TG neurons. In trigeminal sensory nuclei of the brainstem, SCN2B-IR neuronal cell bodies were common in deep laminae of the subnucleus caudalis, and the subnuclei interpolaris and oralis. In the mesencephalic trigeminal tract nucleus, primary sensory neurons also exhibited SCN2B-immunoreactivity. In other regions of trigeminal sensory nuclei, SCN2B-IR cells were very infrequent. SCN2B-IR neuropil was detected in deep laminae of the subnucleus caudalis as well as in the subnuclei interpolaris, oralis and principalis. These findings suggest that SCN2B is expressed by various types of sensory neurons in the TG. There appears to be SCN2B-containing pathway in the TG and trigeminal sensory nuclei.     

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.     

Sensory innervation of the gastro-intestinal junction: new electrophysiological, histological and histochemical data]

The sensory innervation of the small intestine was studied in the cat with electrophysiological, histological and histochemical techniques. Thanks to the histochemical technique (peroxydase method) the exact number and proportion of splanchnic and vagal fibres was determined : the latter being about 9 times more numerous than the former. On the other hand the exact position of the corresponding cells was defined in the nodose and spinal ganglia by means of the previous technique and the microelectrophysiological method (recording of single units into the ganglia with extracellular glass microelectrodes). The splanchnic neurones were found in the T9, T10 and T11 ganglia whereas the vagal ones were chiefly located in the lower half of the nodose ganglia. The histological studies using electronic microscope showed many non-medullated endings, which were often found beneath the epithelium and in the lamina propria of the villi close to the blood vessels. This result is certainly the proof that numerous receptors (mechanoreceptors, chemoreceptors and even thermoreceptors do exist in the small intestine.     

Differential distribution of vanilloid receptors in the primary sensory neurons projecting to the dorsal skin and muscles

We examined transient receptor potential (TRP) V1 and TRPV2 expression in calcitonin gene-related peptide (CGRP) positive (+) primary sensory neurons projecting to the skin and skeletal muscles of the rat dorsum. Among the dorsal root ganglia at the levels from C2 to Th1, 34.9% of neurons projecting to the skin were positive for CGRP, and 32.6% or 21.6% of neurons projecting to the trapezius muscle or the longissimus muscle were positive for CGRP. Of the small CGRP+ neurons projecting to the skin, 53.5% were positive for TRPV1, 11.6% were positive for TRPV2. Of the small CGRP+ neurons projecting to the trapezius or the longissimus, 53.1 or 53.2% were positive for TRPV1, 8.8 or 8.3% were positive for TRPV2, respectively. In the periphery, 29.3% of CGRP+ nerve fibers were positive for TRPV1 in the skin, whereas 65.0 or 59.8% were positive in the trapezius or the longissimus. Therefore, the present study showed that the percentage of CGRP+ neurons projecting to the trapezius is higher than that to the longissimus, and that the co-localization percentage of CGRP and TRPV1 on the sensory nerves was also higher in the trapezius than in the longissimus and the skin.     

Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT- 3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross- sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.     

Functional interaction between transforming growth factor alpha and capsaicin-sensitive sensory neurons in the rat stomach

BACKGROUND AND AIMS: Transforming growth alpha (TGFalpha) and sensory neurons have been shown to promote gastric mucosal protection and healing. Aims were to examine in vitro interactions between gastric sensory neurons, the sensory neuropeptide calcitonin gene-related peptide (CGRP), and TGFalpha. METHODS: Gastric mucosal/submucosal tissue fragments from Sprague-Dawley (SD) rats were incubated in short-term (30 min) culture. Peptide release into media and TGFalpha tissue content were measured by radioimmunoassay. RESULTS: TGFalpha (1 x 10(-8) to 1 x 10(-6) M) caused dose-dependent stimulation of CGRP release. Maximal CGRP release (+87%) was observed with 1 x 10(-6) M TGFalpha: 28.6+/-3.8 vs. control of 15.5+/-2.7 pg/g tissue; P<0.05. Both CGRP (1 x 10(-7) to 1 x 10(-5) M) and capsaicin (1 x 10-(8) to 1 x 10(-6)M) significantly inhibited basal TGFalpha release in a dose-dependent fashion that ranged from -20% to -39%. In contrast, capsaicin-induced sensory denervation caused significant increases in both basal TGFalpha release and TGFalpha tissue content. CONCLUSION: Function interactions between TGFalpha and gastric sensory neurons are suggested by the observations that (1) TGFalpha stimulated CGRP release from gastric sensory neurons; (2) CGRP and acute capsaicin treatment inhibited TGFalpha release and; (3) capsaicin-induced sensory denervation caused significant increases in both gastric TGFalpha basal release and tissue content.     

实验性结肠炎对脊髓传入神经元中降钙素基因相关肽和香草酸受体1表达的调节

本文旨在探讨肠道局部炎症对脊髓肠道感觉传入神经通路的近期及远期效应,应用三硝基苯磺酸(trinitrobenzenesulfonic acid,TNBS)建立大鼠结肠炎动物模型,用DiI(3)逆行神经标记法识别支配肠道炎症部位的脊髓背根神经节(dorsalrootganglia,DRG)神经元,通过肉眼观察、平均组织损伤评分及髓过氧化物酶活性测定等方法评价肠道组织的炎症反应状态,用免疫组织化学法测定香草酸受体l(vanilloid receptor 1,VRl)和降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在支配结肠炎症部位的DRG神经元中的表达,比较炎症不同阶段(给予TNBS后7、21、42 d)CGRP和VRI阳性神经元的数目.结果显示,炎症急性期(即给予TNBS后7 d)结肠黏膜肉眼可见明显损伤,同时相应DRG中表达CGRP及VRl的神经元增加近2倍[(95.38±9.45)%VS(42.86±5.02)%,(89.23±8.21)%VS(32.54±4.58)%].给予TNBS后21、42 d,肠道炎症反应已完全消退,但表达CGRP及VRl的DRG神经元数目仍明显高于对照组[(86.25±8.21)%,(68.28±7.12)%VS(42.86±5.02)%;(67.22±6.52)%,(56.25±4.86)%VS(32.54±4.58)%].结果提示,肠道局部炎症可以上调支配肠道的脊髓传入神经元中CGRP和VRl的表达,这种异常表达可以持续至肠道炎症反应消退后的一定时间.     

Sensory peptide neurotransmitters mediating mucosal and distension evoked neural vasodilator reflexes in guinea pig ileum

The aim was to determine the role CGRP and/or tachykinins released from sensory neural mechanisms in enteric neural vasodilator pathways. These pathways project through the myenteric plexus to submucosal vasodilator neurons. Submucosal arterioles were exposed in the distal portion of an in vitro combined submucosal-myenteric guinea pig ileal preparation, and dilation was monitored with videomicroscopy. Vasodilator neural reflexes were activated by gently stroking the mucosa with a fine brush or by distending a balloon placed beneath the flat-sheet preparation in the proximal portion. Dilations evoked by mucosal stroking were inhibited 64% by the CGRP 8-37 and 37% by NK3 (SR 142801) antagonists. When the two antagonists were combined with hexamethonium, only a small vasodilation persisted. Balloon distension-evoked vasodilations were inhibited by NK3 antagonists (66%) but were not altered by CGRP 8-37. In preparations in which myenteric descending interneurons were directly activated by electrical stimulation, combined application of CGRP 8-37 and the NK antagonists had no effect. Stimulation of capsaicin sensitive nerves in the myenteric plexus did not activate these vasodilator reflexes. These findings suggest that mucosal-activated reflexes result from the release of CGRP and tachykinins from enteric sensory neurons. Distension-evoked responses were significantly blocked by NK3 antagonists, suggesting that stretch activation of myenteric sensory neurons release tachykinins that activate NK3 receptors on myenteric vasodilator pathways.     

Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells

Summary By use of the indirect immunofluorescence technique the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) has been analyzed in cervical and lumbar dorsal root ganglia of untreated and colchicine-treated rats. In addition, lumbar ganglia were examined 2 weeks after transection of the sciatic nerve. The occurrence of CGRP-positive cells in relation to ganglion cells containing substance P-, somatostatin-, galanin-, cholecystokinin (CCK)-, and vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucin (PHI)-LI has been evaluated on consecutive sections as well as using elution-restaining and double-staining techniques.CGRP-LI was observed in many ganglion cells of all sizes ranging in diameter from 15 m to 65 m. Thus, this peptide occurs also in the large primary sensory neurons. In contrast to the sensory peptides described to date, CGRP-positive cells constituted up to 50% of all and 70% of the medium-sized neurons, thus being the most frequently occurring peptide in sensory neurons so far encountered. Subpulations of CGRP-positive neurons were shown to contain substance P-, somatostatin-, or galanin-LI and some CGRP-positive neurons contained both substance P- and galanin-LI. In fact, most substance P-, somatostatin- and galanin-positive cell bodies were CGRP-immunoreactive. The coexistence analysis further revealed that galanin and substance P often coexisted and that some cells contained both substance P- and somatostatin-LI, whereas no coexistence between galanin and somatostatin has as yet been seen. VIP/PHI-LI was only shown in a few cells in untreated or colchicine-treated rats. However, after transcetion of the sciatic nerve numerous VIP/PHI-positive cells were observed, some of which also contained CGRP-LI.The present results indicate that a CGRP-like peptide is present in a wide range of primary sensory neurons probably not related to specific sensory modalities. Often this peptide coexists with other biologically active peptides. Taken together these findings suggest that CGRP may have a generalized function.     

Unit responses in the anterior and posterior hypothalamus to stimulation of the splanchnic and sciatic nerves and to photic stimulation

By extracellular recording of spike discharges the sensory properties of neurons of the anterior and posterior regions of the cat hypothalamus were studied during stimulation of the splanchnic and sciatic nerves and during photic stimulation. Hypothalamic neurons were shown to be characterized by wide convergence of heterosensory excitation: 68% of spontaneously active hypothalamic neurons responded to somatovisceral and photic stimulation. Some posterior hypothalamic neurons responded to somatovisceral stimulation but not to photic stimulation. Neurons responding only to photic stimulation were found in the anterior hypothalamus; no neurons responding only to visceral stimulation were found in the hypothalamus. Total convergence of somatic and visceral afferentation of neurons of the posterior and anterior hypothalamus was observed. Mostly responses of phasic type were obtained to stimulation of all modalities. The study of the quantitative ratio between responses of excitatory and inhibitory types showed that the former predominate. The principles governing the functional organization of hypothalamic afferent systems are discussed.Academician L. A. Orbeli Institute of Physiology, Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 8, No. 3, pp. 276–282, May–June, 1976.     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.     

Effect of intestinal inflammation on capsaicin-sensitive afferents in the ileum of<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis>-infected mice

In the present study, the effect of intestinal schistosomiasis on the extrinsic sensory innervation of the murine ileum was investigated. Immunocytochemical techniques to localize calcitonin gene-related peptide (CGRP), substance P (SP), and vanilloid receptor 1 (VR1) were combined with retrograde tracing techniques and capsaicin treatment. Neurochemical characterization of extrinsic primary afferent neurons (EPANs) in normal and capsaicin-treated mice, revealed that CGRP and VR1, but not SP, were expressed in extrinsic afferents. Immunocytochemical analysis using the above-mentioned antibodies yielded three different populations of neurons in both dorsal root and nodose ganglia, namely CGRP/--, SP/--, and CGRP/SP-expressing neurons. Retrograde tracing revealed that only CGRP/--expressing neurons projected to the ileum. Intestinal schistosomiasis resulted in an upregulation of the number of CGRP-immunoreactive (ir) nerve fibers in the lamina propria of the villi, coinciding with an increase in mucosal mast cells in acutely and chronically infected animals. In infected animals, mucosal mast cells were found closely associated with a dense mucosal CGRP-ir fiber network. Neonatal capsaicin treatment led to a 70% reduction in the number of mucosal mast cells. In conclusion, the present study provides evidence that CGRP is a valid marker for EPANs in the mouse ileum, which are involved in the recruitment of mucosal mast cells. Morphological evidence is provided of a neuroimmune interaction between mucosal mast cells and EPANs in schistosoma-infected mice.     

Wounds increase activin in skin and a vasoactive neuropeptide in sensory ganglia

Successful healing of skin wounds requires sensory innervation and the release of vasoactive neuropeptides that dilate blood vessels and deliver serum proteins to the wound, and that cause pain that protects from further injury. Activin has been proposed as a target-derived regulator of sensory neuropeptides during development, but its role in the mature nervous system is unknown. While adult skin contains a low level of activin, protein levels in skin adjacent to a wound increase rapidly after an excision. Neurons containing the neuropeptide calcitonin gene-related peptide (CGRP) increased in sensory ganglia that projected to the wounded skin, but not in ganglia that projected to unwounded skin, suggesting that neurons respond to a local skin signal. Indeed, many adult sensory neurons respond with increased CGRP expression to the application of activin in vitro and utilize a smad-mediated signal transduction pathway in this response. A second skin-derived factor nerve growth factor (NGF) also increased in wounded skin and increased CGRP in cultured adult dorsal root ganglia (DRG) neurons but with lower efficacy. Together, these data support the hypothesis that activin made by skin cells regulates changes in sensory neuropeptides following skin injury, thereby promoting vasodilation and wound healing.     

The survival and growth of embryonic proprioceptive neurons is promoted by a factor present in skeletal muscle

To date, the neurotrophic factor requirements of developing sensory neurons have been studied using heterogeneous populations of neurons that innervate a wide variety of different sensory structures. To ascertain the particular neurotrophic factor requirements of different kinds of sensory neurons and to determine whether these requirements are related to the type of sensory receptors innervated, it is necessary to study homogeneous preparations of functionally distinct sensory neurons. For this reason I have studied the influence of a soluble extract of skeletal muscle on the survival and growth of proprioceptive neurons isolated from the trigeminal mesencephalic nucleus (TMN) of the embryonic chick. Explants of the TMN and dissociated glia-free cultures of TMN neurons were established from chick embryos of 10 to 18 days incubation (E10 to E18). Skeletal muscle extract prepared from E18 chick pectoral muscle and enriched for neurotrophic activity by ammonium sulfate fractionation promoted marked neurite outgrowth from explants and substantial survival in dissociated cultures established during the period of natural neuronal death in the TMN. In these latter cultures 70 to 80% of the neurons survived and grew in the presence of the extract compared with less than 2% in control cultures. At later ages, following the period of natural neuronal death, these effects were less marked. The neurotrophic activity of extracts prepared from muscle of different ages increased steadily from E10 to E20 (the oldest muscle studied). The active factor is heat labile, trypsin sensitive, and non-dialyzable, it is neither functionally nor immunochemically related to NGF and it has negligible neurotrophic effect on the predominantly cutaneous sensory neuron population of the trigeminal ganglion. These findings demonstrate that skeletal muscle contains a neurotrophic factor which supports the survival and growth of proprioceptive neurons and suggest that this factor has some specificity among functionally distinct kinds of sensory neurons.     

Sensory nerve induced inflammation contributes to heterotopic ossification

Heterotopic ossification (HO), or bone formation in soft tissues, is often the result of traumatic injury. Much evidence has linked the release of BMPs (bone morphogenetic proteins) upon injury to this process. HO was once thought to be a rare occurrence, but recent statistics from the military suggest that as many as 60% of traumatic injuries, resulting from bomb blasts, have associated HO. In this study, we attempt to define the role of peripheral nerves in this process. Since BMP2 has been shown previously to induce release of the neuroinflammatory molecules, substance P (SP) and calcitonin gene related peptide (CGRP), from peripheral, sensory neurons, we examined this process in vivo. SP and CGRP are rapidly expressed upon delivery of BMP2 and remain elevated throughout bone formation. In animals lacking functional sensory neurons (TRPV1(-/-) ), BMP2-mediated increases in SP and CGRP were suppressed as compared to the normal animals, and HO was dramatically inhibited in these deficient mice, suggesting that neuroinflammation plays a functional role. Mast cells, known to be recruited by SP and CGRP, were elevated after BMP2 induction. These mast cells were localized to the nerve structures and underwent degranulation. When degranulation was inhibited using cromolyn, HO was again reduced significantly. Immunohistochemical analysis revealed nerves expressing the stem cell markers nanog and Klf4, as well as the osteoblast marker osterix, after BMP2 induction, in mice treated with cromolyn. The data collectively suggest that BMP2 can act directly on sensory neurons to induce neurogenic inflammation, resulting in nerve remodeling and the migration/release of osteogenic and other stem cells from the nerve. Further, blocking this process significantly reduces HO, suggesting that the stem cell population contributes to bone formation.     

Skin cell induction of calcitonin gene-related peptide in embryonic sensory neurons in vitro involves activin

Target skin cells induce the neuropeptide calcitonin gene-related peptide (CGRP) in na?ve embryonic dorsal root ganglion (DRG) neurons in vitro, but the molecular basis of that induction is not known. Recombinant activin or bone morphogenetic proteins (BMPs) dramatically increase the number of sensory neurons with CGRP and substance P in vitro (X. Ai et al., 1999, Mol. Cell. Neurosci. 14, 506-518). These experiments were designed to test if activin or BMPs accounted for the CGRP-inductive activity by skin cells. To identify factors from skin that induce CGRP, we developed a bioassay in which embryonic DRG neurons isolated before peripheral target contact in vivo are challenged in vitro with specific factors. Conditioned medium from an embryonic rat skin cell line induced neuronal CGRP expression, and induction was blocked by follistatin, implicating transforming growth factor family members. Immunoblot analysis revealed that the skin cell line medium contained several activin and bone morphogenetic protein moieties. Antibody specific to activin neutralized most of the CGRP-inductive activity in skin conditioned medium. These data indicate that the CGRP-inductive action of skin cells involves activin and establish activin as a candidate regulator of this sensory neuropeptide phenotype during development.     

NPY-, galanin-, VIP/PHI-, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections

Summary The neuronal subpopulations in the cat stellate, lower lumbar and sacral sympathetic ganglia were studied with regard to the cellular distribution of immunoreactivity to tyrosine hydroxylase (TH), acetylcholinesterase (AChE) and various neuronal peptides. Coexistence of neuropeptide Y (NPY)- and galanin (GAL)-like immunoreactivity (LI) was found in a high proportion of the neuronal cell bodies; these cells also contained immunoreactivity to TH, confirming their presumably noradrenergic nature. Some TH- and GAL-immunoreactive principal ganglion cells lacked NPY-LI. Two populations (scattered and clustered) of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-positive cell bodies were found in the sympathetic ganglia studied. The scattered VIP/PHI neurons also contained AChE-LI, calcitonin gene-related peptide (CGRP)-and, following culture, substance P (SP)-LI. The clustered type only contained AChE-LI. In the submandibular and sphenopalatine ganglia, neurons were AChE- and VIP/ PHI-immunoreactive but lacked CGRP- and SP-LI. Many GAL- and occasional TH-positive neurons were found in these ganglia. In the spinal ganglia, single NPY-immunoreactive sensory neuronal cells were observed, in addition to CGRP- and SP-positive neurons. The present results show that there are at least two populations of sympathetic cholinergic neurons in the cat. Retrograde tracing experiments indicate that the scattered type of cholinergic neurons contains four vasodilator peptides (VIP, PHI, CGRP, SP) and provides an important input to sweat glands, whereas the clustered type (containing VIP and PHI) mainly innervates blood vessels in muscles.