Substance P-like Immunoreactivity Research Articles

Mass spectrometric studies on the peptide integrity of substance P and related human tachykinins in human biofluids

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice–immunoassays–also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1–178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

Increased substance P-like immunoreactivities in parabrachial and amygdaloid nuclei in a rat model with masticatory myofascial pain.

This study explores the involvement of substance P (SP) in the parabrachial nucleus (PBN) and central amygdaloid nucleus (CeA) in the nociception-emotion link and of rats with masticatory myofascial pain (MMP) induced by chronic tetanic eccentric muscle contraction. A total of 18 rats were randomly and equally assigned for MMP (MMP group) and sham-MMP induction (sMMP group). MMP was induced by electrical-stimulated repetitive tetanic eccentric contraction of the masseter muscle for 14 consecutive days. Myofascial trigger points in the masseter muscle were identified by palpable taut bands, increased prevalence of endplate noise (EPN), focal hypoechoic nodules on ultrasound and restricted jaw opening. All animals were killed for morphological and SP immunohistochemical analyses. Chronic tetanic eccentric contraction induced significantly thicker masseter muscle confirmed by hypoechogenicity, increased prevalence and amplitudes of EPN, and limited jaw opening. Immunohistochemically, the SP-like positive neurons increased significantly in PBN and CeA of the MMP group. Our results suggested that MMP increases the SP protein levels in PBN and CeA, which play important roles in MMP-mediated chronic pain processing as well as MMP-related emotional processes.

Vagotomy decreases the neuronal activities of medulla oblongata and alleviates neurogenic inflammation of airways induced by repeated intra-esophageal instillation of HCl in guinea pigs.

Neuronal activity in the medulla oblongata and neurogenic inflammation of airways were investigated in a guinea pig model induced by repeated intra-esophageal instillation of hydrochloric acid (HCl) after vagotomy. Unilateral vagotomy was performed in the vagotomy group, while a sham-operation was performed in the sham group. Operation was not conducted in sham control group. Airway inflammation was observed with hematoxylin and eosin (HE) staining. C-fos protein was measured by immunohistochemistry (IHC) and Western blot (WB). Substance P was examined by IHC and enzyme-linked immuno sorbent assay (ELISA). Airway microvascular permeability was detected by evans blue dye (EBD) fluorescence. Inflammation of airway was observed in the trachea and bronchi after chronic HCl perfusion into the lower esophagus, and was alleviated after unilateral vagotomy. C-fos expression in the medulla oblongata was lower in the vagotomy group compared to the sham control and sham groups. Substance P-like immunoreactivity (SP-li), concentration and microvascular leakage in airway were lower in the vagotomy group than that in the other groups. Our results suggest that vagotomy improved neurogenic inflammation of airways and decreased neuronal activities, the afferent nerves and neurons in medulla oblongata may be involved in neurogenic inflammation of airways mediated by esophageal-bronchial reflex.

The sensory trigeminal complex and the organization of its primary afferents in the zebra finch (Taeniopygia guttata).

Our knowledge of the avian sensory trigeminal system has been largely restricted to the principal trigeminal nucleus (PrV) and its ascending projections to the forebrain. Studies addressing the cytoarchitecture and organization of afferent input to the sensory trigeminal complex, which includes both the PrV and the nuclei of the descending trigeminal tract (nTTD), have only been performed in pigeons and ducks. Here we extend such an analysis to a songbird, the zebra finch (Taeniopygia guttata). We describe the cytoarchitecture of the sensory trigeminal complex, the patterns of calbindin-like and substance P-like immunoreactivity, and the organization of afferents from the three branches of the trigeminal nerve and from the lingual branch of the hypoglossal nerve. On the basis of cytoarchitecture and immunohistochemistry, the sensory trigeminal column can be subdivided from caudal to rostral, as in other species, into cervical dorsal horn, subnucleus caudalis, subnucleus interpolaris, subnucleus oralis, and nucleus principalis. The relative positions of the terminal fields of the three trigeminal branches move from medial to lateral in the dorsal horn to dorsomedial to ventrolateral in nTTD, whereas in PrV there is considerable overlap of mandibular and ophthalmic terminal fields, with only a small maxillary input ventrally. The hypoglossal afferents, which terminate medially in the dorsal horn and dorsolaterally in nTTD, terminate in specific cell groups in the dorsolateral nTTDo and in PrV. This work sets the grounds for further analyses of the ascending connections of the nTTD and the afferents from the syrinx to the trigeminal sensory column.

Inflammation enhances Y1 receptor signaling, neuropeptide Y-mediated inhibition of hyperalgesia, and substance P release from primary afferent neurons

Neuropeptide Y (NPY) is present in the superficial laminae of the dorsal horn and inhibits spinal nociceptive processing, but the mechanisms underlying its anti-hyperalgesic actions are unclear. We hypothesized that NPY acts at neuropeptide Y1 receptors in the dorsal horn to decrease nociception by inhibiting substance P (SP) release, and that these effects are enhanced by inflammation. To evaluate SP release, we used microdialysis and neurokinin 1 receptor (NK1R) internalization in rat. NPY decreased capsaicin-evoked SP-like immunoreactivity in the microdialysate of the dorsal horn. NPY also decreased non-noxious stimulus (paw brush)-evoked NK1R internalization (as well as mechanical hyperalgesia and mechanical and cold allodynia) after intraplantar injection of carrageenan. Similarly, in rat spinal cord slices with dorsal root attached, [Leu31, Pro34]-NPY inhibited dorsal root stimulus-evoked NK1R internalization. In rat dorsal root ganglion neurons, Y1 receptors colocalized extensively with calcitonin gene-related peptide (CGRP). In dorsal horn neurons, Y1 receptors were extensively expressed and this may have masked the detection of terminal co-localization with CGRP or SP. To determine whether the pain inhibitory actions of Y1 receptors are enhanced by inflammation, we administered [Leu31, Pro34]-NPY after intraplantar injection of complete Freund’s adjuvant (CFA) in rat. We found that [Leu31, Pro34]-NPY reduced paw clamp-induced NK1R internalization in CFA rats but not uninjured controls. To determine the contribution of increased Y1 receptor-G protein coupling, we measured [35S]GTPγS binding simulated by [Leu31, Pro34]-NPY in mouse dorsal horn. CFA inflammation increased the affinity of Y1 receptor G-protein coupling. We conclude that Y1 receptors contribute to the anti-hyperalgesic effects of NPY by mediating the inhibition of SP release, and that Y1 receptor signaling in the dorsal horn is enhanced during inflammatory nociception.

Botulinum toxin type a therapy in migraine: preclinical and clinical trials.

BackgroundBotulinum toxin type A (BTX-A) has been reported to be effective for the therapy for migraine. The purpose of this study was to investigate the effect of BTX-A on the immunoreactive levels of calcitonin gene-related peptide (CGRP) and substance P (SP) in the jugular plasma and medulla oblongata of migraine in rats induced by nitroglycerin (NTG), and then to evaluate and compare the effectiveness of fixed (muscle)-sites and acupoint-sites injection of BTX-A for migraine therapy of patients in a randomly controlled trial extending over four months.Materials and MethodsRats with NTG-induced migraine were subcutaneously injected with vehicle or BTX-A (5 U/kg or 10 U/kg bodyweight). CGRP- and SP-like immunoreactivity (CGRP-LI and SP-LI) were determined by radioimmunoassay. In clinical trials, sixty patients respectively received BTX-A (2.5 U each site, 25 U per patient) at fixed-sites (group F, n = 30) including occipitofrontalis, corrugator supercili, temporalis and trapezius or at acupoint-sites (group A, n = 30) including EX-HN3, EX-HN5, GV20, GB8, GB20 and BL10.ResultsLocal BTX-A injection suppressed NTG-induced CGRP-LI and SP-LI levels in jugular plasma and oblongata. BTX-A injection for both groups with migraine significantly reduced the attack frequency, intensity, duration and associated symptoms. The efficacy of BTX-A for migraine in group A (93% improvement) was more significant than that in group F (83% improvement) (P < 0.01).ConclusionsThe evidence that BTX-A decreases NTG-induced CGRP-LI and SP-LI levels in trigeminovascular system suggests that BTX-A attenuates migraine by suppression of neuropeptide release. BTX-A injections for migraine at acupoint-sites and fixed-sites are effective. Acupoint-sites BTX-A administration shows more efficacy for migraine than fixed-sites application.

Substance P as a neuronal factor in the enteric nervous system of the porcine descending colon in physiological conditions and during selected pathogenic processes

The present investigation pertains to changes in substance P-like immunoreactive (SP-LI) nerve structures of the enteric nervous system (ENS) in the porcine descending colon, caused by chemically-induced inflammation and nerve injury (axotomy). The distribution pattern of SP-LI structures was studied using the double immunofluorescence technique in the myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses, as well as in the circular muscle and mucosal layers. Under physiological conditions, SP-LI neurons have been shown to constitute 4.13 ± 0.24%, 3.36 ± 0.26%, and 7.92 ± 0.16% in the MP, OSP, and ISP, respectively. Changes in SP-immunoreactivity depended on the pathological factor studied. The numbers of the SP-LI perikarya amounted to 7.89 ± 0.34, 5.56 ± 0.30, and 19.96 ± 0.57 in chemically-induced colitis, and 4.28 ± 0.13%, 7.18 ± 20%, and 11.62 ± 0.48% after axotomy in MP, OSP, and ISP, respectively. The both studied processes generally resulted in an increase in the number of SP-LI nerve fibers in the circular muscle and mucosal layers. The obtained results suggest that SP-LI nerve structures of the ENS may participate in various pathological processes in the porcine descending colon and exact functions of SP probably depend on the type of the pathological factor.

Neuropeptide levels in Dercum's disease (adiposis dolorosa)

Dercum's disease (adiposis dolorosa) is characterised by adiposity and chronic pain in the adipose tissue. It has been proposed that conditions encompassing chronic pain have altered concentrations of neuropeptides involved in pain transmission. The aim of this investigation was to examine whether patients with Dercum's disease have abnormal concentrations of different neuropeptides. In cerebrospinal fluid (CSF) and in plasma (P) from 53 patients with Dercum's disease substance P-like immunoreactivity (SP-LI), neuropeptide Y-like immunoreactivity (NPY-LI), β-endorphin-like immunoreactivity (β-END-LI), calcitonin gene-related peptidelike immunoreactivity (CGRP-LI), met-enkephalin-like immunoreactivity (m-ENK-LI), vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI), somatostatin (SOM-LI), γ2-melanocyte-stimulating hormone-like immunoreactivity (γ2-MSH-LI), and dynorphin-like immunoreactivity (DYN-LI) were measured. Three of the substances were also measured in a control group. The CSF concentration of SP was statistically significantly lower in the Dercum group than in the control group, whereas NPY-LI and b-END-LI were borderline statistically significantly lower and higher, respectively, in Dercum patients compared to controls. Compared with reference values, CSF-MSH-LI levels were slightly elevated and CSF-NPY-LI levels were slightly lowered in the Dercum group. The other substances in both CSF and plasma were within the reference values with a high degree of statistical significance. In conclusion, altered levels of neuropeptides that have previously been seen in different pain conditions cannot clearly be demonstrated in Dercum's disease.

Cerebrospinal Fluid Substance P-Like Immunoreactivity Correlates with Aggression in Personality Disordered Subjects

Neurochemical studies have pointed to a modulatory role in human aggression for a variety of central neurotransmitters; some seem to play an inhibitory role, whereas others seem to play a facilitory role in the modulation of aggression. Laboratory animal studies of substance P suggest a facilitory role for this undecapeptide in the modulation of aggression, but no studies of substance P have yet been reported with regard to human aggression. Basal lumbar cerebrospinal fluid samples were obtained from 38 physically healthy subjects with personality disorder (PD) and substance P-like immunoreactivity was measured and correlated with measures of aggression and impulsivity. The cerebrospinal fluid substance P-like immunoreactivity levels were directly correlated with a composite measure of aggression and, more specifically, with Buss-Durkee Aggression. No correlation was seen with any measure of impulsivity or of general dimensions of personality. These data suggest a direct relationship between central nervous system substance P containing neural circuits and aggression in human subjects. This finding adds to the complex picture of the central neuromodulatory role of impulsive aggression in human subjects.

Effects of gentamicin on guinea pig vestibular ganglion function and on substance P and neuropeptide Y

Previous studies have demonstrated that following intratympanic gentamicin application in the guinea pigs, vestibular evoked myogenic potentials (VEMPs) were absent regardless of stimulation mode using either air-conducted sound (ACS) stimuli or galvanic vestibular stimulation (GVS). Ultrastructurally, both type I hair cells and their calyx terminals were distorted in the saccular macula. However, little is known about the toxic effects of gentamicin on the vestibular ganglion (VG). In this study, absent ACS- and GVS-VEMPs were noted in all the gentamicin-treated ears (100%), which were confirmed by the substantial loss of sensory hair cells in the saccular macula. Moreover, dramatic up-regulation of growth associated protein-43 (GAP-43) expression was detected in the ipsilateral VG neurons. The mean percentage of substance P-like immunoreactive (SP-LI) neurons in the treated VG (81.8 ± 1.9%) was significantly higher than that in the control VG (68.6 ± 3.3%). Conversely, the mean percentage of neuropeptide Y-like immunoreactive (NPY-LI) neurons in the treated VG (13.7 ± 3.8%) was dramatically lower than that in the control VG (49.0 ± 3.8%). Double labeling results shown 82% of SP-LI and 16% of NPY-LI neurons coexpressed with GAP-43, suggested that SP accumulating coincided with NPY decreasing in regenerating VG neurons after gentamicin treatment. Overall, the changes in SP and NPY expression in VG neurons after gentamicin treatment were like to those in the superior cervical ganglion following sympathectomy.

P.1.c.063 Alpha-1-noradrenergic receptor blockade in OCD: an open label add-on study with prazosin

Glutamate participates in the regulation of secretion of several neuropeptides, including substance P (SP). Glutamate acts through ionotropic (iGluR) and metabotropic (mGluR) receptors. We have investigated whether glutamate receptor agonists and antagonists could affect SP release from the arcuate nucleus and the median eminence (ARC/ME). An increase in SP-like immunoreactivity (SP-LI) release from ARC/ME was induced by glutamate and N-methyl-d-aspartate (NMDA). This increase was prevented by d-(−)-2-amino-5-phosphono pentanoic acid (DAP5) (0.1 mM), a specific NMDA antagonist and by (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (0.1 mM), a selective antagonist of group I mGluR. The selective non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3(1H-4H)-dione (DNQX) (0.1 mM) and (RS)-α-methyl-4-tetrazolylphenylglycine (MTPG) (0.1 mM), a group II and III mGluRs antagonist, did not affect the stimulatory effect of glutamate. A group I selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) induced a significant increase in SP-LI release. Supporting the participation of nitric oxide (NO) in the effect of glutamate on SP-LI release, NAME (0.5 mM), a NO synthase inhibitor, reduced the glutamate-induced increase in SP-LI release from ARC/ME. Similarly, glutamate did not induce an increase in SP-LI release in the presence of meloxicam (0.1 mM) (a cyclooxygenase-2 (COX-2) specific inhibitor) indicating that prostaglandins production may also be involved in the glutamate effect. These data indicate that glutamate increases SP-LI release from the ARC/ME by acting through NMDA and group I mGluRs in the male rat. This stimulatory effect could be mediated by nitric oxide and prostaglandin production.

Neuron-specific enolase-like immunoreactivity in human Leydig cells

Using the peroxidase anti-peroxidase immunocytochemical technique, neuron-specific enolase (NSE)-like immunoreactivity (NSE-LI) was revealed in Leydig cells of adult human testes at the light microscopic level. Differences in the NSE staining intensity were observed between the individual Leydig cells, separate cell groups within a testis and between the testes of individual patients. Together with the already established substance P-like immunoreactivity (SP-LI), the results obtained provided further evidence for the possible neuroectodermal origin of human Leydig cells and their presumable relation to the APUD- or the Diffuse Neuroendocrine System (DNES).

Prenatal Exposure to Nicotine Affects Substance P and Preprotachykinin-A mRNA Levels in Newborn Rat

Prenatal nicotine exposure influences neuronal development including effects on several neurotransmitter systems. It also attenuates the ventilatory response to hypoxia, known to require a functional substance P-ergic system. Previous studies have shown that nicotine increases the risk for sudden infant death syndrome (SIDS) by 4-fold, and that SIDS-victims have elevated brainstem levels of substance P. We, therefore, studied the effect of prenatal nicotine exposure on the levels of substance P-like immunoreactivity by RIA in the brain in newborn rat pups. The expression of the substance P precursor preprotachykinin A mRNA was also determined by real-time reverse transcriptase-polymerase chain reaction in carotid body, in petrosal/jugular and trigeminal ganglia, in cervical and lumbar dorsal root ganglia, and in the brainstem. We found that prenatal nicotine exposure increased levels of substance P-like immunoreactivity in the brainstem without changing levels in other parts of the brain or in the adrenals. Furthermore, mRNA levels were increased in the carotid bodies and in the petrosal ganglia, in contrast to the decreased levels in the cervical dorsal root ganglia. We conclude that nicotine causes alterations in the substance P-ergic system in the brainstem, possibly linked to the increased risk for SIDS after prenatal nicotine exposure.

Responses of limbic and extrapyramidal substance P systems to nicotine treatment.

Neuropeptides are linked to the psychopathology of stimulants of abuse, principally through dopamine mechanisms. Substance P (SP) is one of these neuropeptides and is associated with both limbic and extrapyramidal dopaminergic pathways and likely contributes to the pharmacology of these stimulants. The effects of nicotine on these dopamine systems have also been extensively studied; however, its effects on the associated SP pathways have received little attention. In the present study, we elucidated the effects of nicotine treatment on limbic and extrapyramidal SP systems by measuring changes in associated SP tissue concentrations. Male Sprague-Dawley rats received (+/-)nicotine 4.0 mg/kg/day (0.8 mg/kg, intraperitoneally; five injections at 2-h intervals) in the presence or absence of selective dopamine D1 and D2 receptor antagonists or a nonselective nicotinic acetylcholine receptor antagonist. The nicotine treatment significantly but temporarily decreased substance P-like immunoreactivity (SPLI) content in the ventral tegmental area (VTA) and substantia nigra 12-18 h after drug exposure. The nicotine-mediated changes in SPLI were selectively blocked by pretreatment with mecamylamine as well as a dopamine D1, D2, or both receptor antagonists. Other brain areas that also selectively demonstrated nicotine-related declines in SPLI content included prefrontal cortex, the nucleus accumbens shell, and the very posterior caudate. These findings indicate that some limbic and basal ganglia SP systems are significantly affected by exposure to nicotine through processes mediated by nicotinic and dopaminergic receptors, suggesting a role for SP pathways in nicotine's limbic and extrapyramidal effects.

Trophic effects of keratinocytes on the axonal development of sensory neurons in a coculture model

The epidermis, the outermost structure of the skin, fulfils important roles as a physical barrier between the organism and its environment and as a neuroendocrine, immune and sensory organ. It is innervated by unmyelinated sensory fibres conveying nociceptive and thermoceptive information. Little is known concerning the functional interactions between these sensory fibres and the keratinocytes, which constitute 95% of the epidermal cells. We have developed a coculture model of primary rat sensory neurons and keratinocytes, as well as of equivalent cell-lines: ND7-23 neurons and A431 keratinocytes. We show that primary dorsal root ganglion neurons survive well in a standard keratinocyte reference medium containing a low concentration of calcium, but fail to extend axons. However, when neurons are cocultured with keratinocytes, axonal outgrowth is strongly stimulated. The use of a Transwell culture system indicated that the stimulation of axonal growth depends on a soluble factor secreted by keratinocytes. Axon outgrowth was also induced by nerve growth factor or brain-derived neurotrophic factor, but not by neurotrophin 3 or glial cell-derived neurotrophic factor. Neurons cocultured with keratinocytes did not change their responses to ATP, capsaicin or high potassium solution, as measured by calcium imaging. The trophic effect of keratinocytes concerned essentially a population of medium-sized (17-25 microm) neurons, some of which expressed substance P-like immunoreactivity and responded to capsaicin. Our preparation, in which cells are maintained at low external calcium concentration, could represent a useful in vitro model for characterizing the effect of skin-derived guidance and trophic factors.

A histologically derived stereotaxic atlas and substance P immunohistochemistry in the brain of the least shrew ( Cryptotis parva) support its role as a model organism for behavioral and pharmacological research

Chemotherapy is an effective treatment but difficult to tolerate due to side effects like vomiting. Studies on the etiology of chemotherapy-related emesis have implicated brainstem nuclei and the neurotransmitter substance P, among other substrates. Since rodents do not vomit, other species have been necessary as alternative models of chemotherapy-induced emesis. Of these, the least shrew ( Cryptotis parva) has proven valuable due to its small size, hardiness, and close phylogenetic relationship with primates. However, very little neuroanatomical data on C. parva exist. We used histological and immunohistochemical techniques to provide neuroanatomical data to help validate C. parva as a model organism, especially for emesis research. Brains were sectioned and stained for Nissl substance or myelin, or immunofluorescently labeled for substance P. Sections were photographed, traced, and reconstructed with standardized zero points, and these data used to create a stereotaxic atlas. The brain of C. parva was similar to but smaller than other mammalian brains, with the cerebellum and hippocampus demonstrating the biggest differences. Differences appeared to be related to the small size of the brain and the metabolic compromises required of such a small mammal. Substance P-like immunoreactivity (SPL-IR) was semiquantitatively mapped, and correlated very well with SPL-IR observed in other species. Dense SPL-IR areas included the periaqueductal grey, trigeminal nuclei, dorsal raphe, and emesis-related brainstem nuclei including the area postrema and solitary tract nucleus. These data demonstrate that the anatomical differences between C. parva and other mammals will not preclude its use as a model organism.

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

The expression of glial fibrillary acidic protein (GFAP) by perivascular cells of many mammalian organs suggests an as yet unknown function of this intermediate filament protein in the maintenance of homeostasis and vascular permeability at the blood-tissue interface. Although a similar situation may exist at the air-tissue interface, the cellular distribution of GFAP in skin tissue has never been demonstrated. To approach this issue, we have employed immunofluorescence and Western blotting techniques to detect GFAP in skin sections of young and adult humans, normal rodents, and two types of mutant mice, as well as in rat lung sections, and in cultured human keratinocytes and fibroblasts. Colocalization with antigens known to be associated with GFAP in other tissues was also tested. Epidermal and hair follicle keratinocytes and dermal fibroblasts showed distinct staining for GFAP as well as colocalization with alpha-actin, metallothionein, and antigens of the class-II major histocompatibility complex (MHC II). GFAP was also identified in rat alveolar fibroblasts which, in common with keratinocytes, form part of the air-tissue interface. GFAP was upregulated together with MHC II in nude mice but was barely detectable in the skin of non-obese diabetic severe combined immunodeficiency mice, suggesting a possible involvement in antigen-presenting functions. The intriguing distribution of a common set of antigens both in certain cells of the integumentary system and at the blood-tissue interfaces of internal organs suggests the involvement of these proteins in universal mechanisms controlling tissue homeostasis and protection.

Localization and expression of substance P in transgenic mice overexpressing human APP751 with the London (V717I) and Swedish (K670M/N671L) mutations

Substance P-like immunoreactivity (-LI) is found in neuritic plaques, and is reduced in patients suffering from Alzheimer disease (AD). In this study, we examined the distribution and expression of substance P in transgenic mice overexpressing human amyloid precursor protein (hAPP) APP751 with the London (V717I) and Swedish (K670M/N671L) mutations. Immunohistochemistry was performed to localize substance P- and glial fibrillary acidic protein-LI by confocal microscopy. In hAPP transgenic mice, the number of β-amyloid plaques significantly increased from 6 to 12 months. About 5% of β-amyloid plaques were substance P-immunoreactive. In transgenic mice, the morphology of substance P-immunoreactive structures changed by consisting of swollen and dystrophic neurites mostly associated with β-amyloid plaques. The overall localization and the relative substance P densities were not different between wild type and transgenic mice at 6 and 12 months. At month 12, a dramatic change in the distribution pattern of substance P-LI was observed as it was now expressed in a high number of reactive astrocytes. This expression of substance P in astrocytes was mainly found in the hippocampal formation and thalamic nuclei with a preferential association with β-amyloid plaques, whereas in cortical regions only faintly substance P-immunoreactive astrocytes were observed. This study indicates that substance P undergoes complex changes in this animal Alzheimer disease model. Future experiments including substance P antagonists are necessary to further explore the interaction between β-amyloid deposits and substance P.

First localization and biochemical identification of chromogranin B- and secretoneurin-like immunoreactivity in the fetal human vagal/nucleus solitary complex

The human vagal/nucleus solitary complex is a primary visceral relay station and an integrative brain stem area which displays a high density of chromogranin B- and secretoneurin-like immunoreactivity. In this study, we localized and biochemically identified these proteins during prenatal development. At prenatal week 11, 15, 20 and 37, we performed a chromatographic analysis to identify the molecular forms of PE-11, a peptide within the chromogranin B sequence, and secretoneurin, a peptide within secretogranin II. Their localization was studied with immunocytochemistry, and was compared to that of substance P which is well established as a functional neuropeptide in the vagal/nucleus solitary complex. At prenatal week 11, chromogranin B-, secretoneurin- and substance P-like immunoreactivities were detected consisting of varicosities, varicose fibers and single cells. At the same time, PE-11 and secretoneurin appeared as a single peak in chromatographic analysis. Prohormone convertases PC1- and PC2-like immunoreactivities were also present at week 11. In general, the density for each peptide increased during later fetal stages with the highest density at week 37. These results demonstrate that each chromogranin peptide is expressed during human fetal life in neurons of the vagal/nucleus solitary complex indicating that these peptides could be important during prenatal development.

Deletion of the neuropeptide Y Y1 receptor affects pain sensitivity, neuropeptide transport and expression, and dorsal root ganglion neuron numbers

Neuropeptide Y has been implicated in pain modulation and is substantially up-regulated in dorsal root ganglia after peripheral nerve injury. To identify the role of neuropeptide Y after axotomy, we investigated the behavioral and neurochemical phenotype of neuropeptide Y Y1 receptor knockout mice with focus on dorsal root ganglion neurons and spinal cord. Using a specific antibody Y1 receptor immunoreactivity was found in dorsal root ganglia and in dorsal horn neurons of wild-type, but not knockout mice. The Y1 receptor knockout mice exhibited a pronounced mechanical hypersensitivity. After sciatic nerve axotomy, the deletion of Y1 receptor protected knockout mice from the axotomy-induced loss of dorsal root ganglion neurons seen in wild-type mice. Lower levels of calcitonin gene-related peptide and substance P were identified by immunohistochemistry in dorsal root ganglia and dorsal horn of knockout mice, and the axotomy-induced down-regulation of both calcitonin gene-related peptide and substance P was accentuated in Y1 receptor knockout. However, the transcript levels for calcitonin gene-related peptide and substance P were significantly higher in knockout than in wild-type dorsal root ganglia ipsilateral to the axotomy, while more calcitonin gene-related peptide- and substance P-like immunoreactivity accumulated proximal and distal to a crush of the sciatic nerve. These results indicate that the deletion of the Y1 receptor causes increased release and compensatory increased synthesis of calcitonin gene-related peptide and substance P in dorsal root ganglion neurons. Together, these findings suggest that, after peripheral nerve injury, neuropeptide Y, via its Y1 receptor receptor, plays a key role in cell survival as well as in transport and synthesis of the excitatory dorsal horn messengers calcitonin gene-related peptide and substance P and thus may contribute to pain hypersensitivity.