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Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord, leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA was very short, resulting in half-lives of 1.9 and 2.2 min respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK 1-26 , NKA, NKA 2-10 , NKA 3-10 , NKA 5-10 and NKA 6-10 , which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA, providing strong evidence that proprotein convertase is involved in C-terminal processing of NPK in the spinal cord, leading to the formation of NKA., (Copyright © 2018 John Wiley & Sons, Ltd.)

In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.

Objective: To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium., Design: Laboratory study., Setting: University research laboratories and academic hospital., Patient(s): Women undergoing hysterectomy for symptomatic leiomyomas., Intervention(s): Quantitative polymerase chain reaction, immunohistochemistry and Western blot., Main Outcome Measure(s): Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium., Result(s): TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells., Conclusion(s): These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The naso-ocular interaction in allergic rhinoconjunctivitis is well recognized from epidemiological, clinical, and experimental observations. The precise mechanisms remain incompletely understood. A new mouse model of allergic rhinoconjunctivitis was used to investigate the contribution of mast cells and trigeminal ganglia activation to conjunctival (conj.) inflammation after nasal allergen provocation., Methods: Sensitized mice were exposed to ovalbumin (OVA) via the nose and/or conjunctiva, and conj. homogenates were analyzed for histamine and substance P (using ELISA) and by eosinophil peroxidase (EPO) and beta-hexosaminidase assays. The conj. effects of nasal allergen deposition were compared with those induced by the mast cell activator C48/80 and with pretreatment of the mast cell stabilizer ketotifen or the transient receptor potential channel receptor (TRP) agonist capsaicin. Protachykinin 1 (TAC1) expression was quantified in the trigeminal ganglia using real time polymerase chain reaction., Results: At 1 hour after nasal application of OVA, increased conj. levels of beta-hexosaminidase (0.68 ± 0.03 nm versus 0.56 ± 0.02 nm; p = 0.02), histamine (751.1 ± 52.17 ng/mL versus 546.3 ± 76.91 ng/mL; p = 0.05), and EPO (0.66 ± 0.09 nm versus 0.37 ± 0.03 nm; p = 0.02) were detected compared with saline. Higher levels of TAC1 expression were found in the trigeminal ganglia at 24 hours after OVA application (1326 ± 255 versus 687.5 ± 90.77 TAC1/beta-actin; p = 0.04). Nasal challenge with C48/80 increased substance P and beta-hexosaminidase levels in the conjunctiva, as well as TAC1 expression. Pretreatment with ketotifen resulted in lower levels of substance P as well as TAC1 expression. Destruction of sensory nerves in the nose by capsaicin reduced the OVA-induced conj. levels of substance P, histamine, and beta-hexosaminidase., Conclusion: Nasal allergen deposition in sensitized mice induced trigeminal TAC1 expression and conj. mast cell degranulation. These data represent a significant step forward in understanding the close interaction between nasal and conj. inflammation in allergy.

A role for enhanced peptidergic transmission, either opioidergic or not, has been proposed for the generation of l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) on the basis of in situ hybridization studies showing that striatal peptidergic precursor expression consistently correlates with LID severity. Few studies, however, have focused on the actual peptides derived from these precursors. We used mass-spectrometry to study peptide profiles in the putamen and globus pallidus (internalis and externalis) collected from 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine treated macaque monkeys, acutely or chronically treated with l-DOPA. We identified that parkinsonian and dyskinetic states are associated with an abnormal production of proenkephalin-, prodynorphin- and protachykinin-1-derived peptides in both segments of the globus pallidus. Moreover, we report that peptidergic processing is dopamine-state dependent and highly structure-specific, possibly explaining the failure of previous clinical trials attempting to rectify abnormal peptidergic transmission., (© 2013.)

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.

Animal models are widely used to perform basic scientific research in pain. The rodent chronic constriction injury (CCI) model is widely used to study neuropathic pain. Animals were tested prior and after CCI surgery using behavioral tests (von Frey filaments and Hargreaves test) to evaluate pain. The brain and the lumbar enlargement of the spinal cord were collected from neuropathic and normal animals. Tachykinin related peptides were analyzed by high performance liquid chromatography quadrupole ion trap mass spectrometry. Our results reveal that the β-tachykinin₅₈₋₇₁, SP and SP₃₋₁₁ up-regulation are closely related to pain behavior. The spinal β-tachykinin₅₈₋₇₁, SP and SP₃₋₁₁ concentrations were significantly up-regulated in neuropathic animals compared with normal animals (p<0.001; p<0.001 and p<0.05, respectively). In contrast, the spinal SP5₅₋₁₁ concentration in neuropathic animals revealed a significant down-regulation compared with normal animals (p<0.05). The brain β-tachykinin₅₈₋₇₁ and SP concentrations were significantly up-regulated (p<0.05 and p<0.001, respectively). Interestingly, no significant concentration differences were observed in the spinal cord and brain for NKA, β-tachykinin₅₈₋₇₁, SP₁₋₇ and SP₆₋₁₁ (p>0.05). The β-tachykinin₅₈₋₇₁, SP and C-terminal SP metabolites could potentially serve as biomarkers in early drug discovery., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Olfaction is in many species the most important sense, essential for food search, mate finding, and predator avoidance. Butterflies have been considered a microsmatic group of insects that mainly rely on vision due to their diurnal lifestyle. However, an emerging number of studies indicate that butterflies indeed use the sense of smell for locating food and oviposition sites. To unravel the neural substrates for olfaction, we performed an anatomical study of 2 related butterfly species that differ in food and host plant preference. We found many of the anatomical structures and pathways, as well as distribution of neuroactive substances, to resemble that of their nocturnal relatives among the Lepidoptera. The 2 species differed in the number of one type of olfactory sensilla, thus indicating a difference in sensitivity to certain compounds. Otherwise no differences could be observed. Our findings suggest that the olfactory system in Lepidoptera is well conserved despite the long evolutionary time since butterflies and moths diverged from a common ancestor.

Objective: To test the hypothesis that experimental occlusal interferences increase the nerve growth factor (NGF) levels in periodontal tissues and cause an up-regulation of preprotachykinin-A (PPTA) mRNA., Background: NGF is related to hyperalgesia and inflammation. PPTA mRNA, a primer of substance P, is a possible factor in the aetiology of pain., Methods: Experimental interferences were created by placing inlays in the right maxillary molars of 15 dogs. The right side molars formed the experimental group. The left side molars served as controls. Three dogs with cavities prepared without changing the occlusion formed a sham group. The dogs in the first group were sacrificed, 3 at each time, after 3, 7, 14, 30, and 60 days. The sham group was sacrificed after 14 days. The levels of NGF in periodontal tissues and PPTA mRNA in the trigeminal ganglions were detected by ELISA and TR-PCR. Comparisons were made with paired t-tests and a multivariate MANOVA test., Results: On all measurement days, there were higher levels of NGF mRNA, PPTA mRNA, and NGF on the experimental than on the control side in 14 of 15 comparisons and in the sham group. NGF production in periodontium was time-dependent. No differences in NGF protein levels were observed between the control and the sham groups., Conclusion: The results which need confirmation in further tests are of clinical interest. They indicate that occlusal experimental interferences may be an etiologic factor in oral facial pain by increasing mRNA and NGF protein levels in the periodontal tissues., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The central complex is a major integrative region within the insect brain with demonstrated roles in spatial orientation, the regulation of locomotor behavior, and sound production. In the hemimetabolous grasshopper, the central complex comprises the protocerebral bridge, central body (CB), ellipsoid body, noduli, and accessory lobes, and this modular organization develops entirely during embryogenesis. From a biochemical perspective, a range of neuroactive substances has been demonstrated in these modules of the adult central complex, but little is known about their developmental expression. In this study, we use matrix-assisted laser desorption/ionization-imaging mass spectrometry on single brain slices to confirm the presence of several peptide families (tachykinin, allatostatin, periviscerokinin/pyrokinin, FLRFamide, and neuropeptide F) in the adult central complex and then use immunohistochemistry and histology to examine their developmental expression, together with that of the indolamin serotonin, and the endogenous messenger nitric oxide (NO; via its synthesizing enzyme). We find that each neuromodulator is expressed according to a unique, stereotypic, pattern within the various modules making up the central complex. Neuropeptides such as tachykinin (55%) and allatostatin (65%), and the NO-synthesizing enzyme diaphorase (70%), are expressed earlier during embryonic development than the biogenic amine serotonin (80%), whereas periviscerokinin-like peptides and FLRFamide-like peptides begin to be expressed only postembryonically. Within the CB, these neuroactive substances are present in tangential projection neurons before they appear in columnar neurons. There is also no colocalization of serotonin-positive and peptide-positive projections up to the third larval instar during development, consistent with the clear dorsoventral layering of the neuropil we observe. Our results provide the first neurochemical fingerprint of the developing central complex in an hemimetabolous insect., (© 2010 Wiley-Liss, Inc.)

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antannal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). We show here that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using two-photon microscopy, we found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. We detect opposite behavioral phenotypes after reduction and over expression of DTKR in ORNs. Our findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe.

The pars tuberalis (PT) is a distinct subdivision of the anterior pituitary gland that plays a central role in regulating seasonal prolactin release. In sheep, there is compelling evidence that seasonal changes in light, transformed into a melatonin signal, are interpreted by the PT to modulate the release of a factor which affects prolactin release. The identity of this factor(s) is unknown but has been preemptively called 'tuberalin'. In the present study, we report on an initial immunocytochemical investigation where we have identified that many ovine PT cells are immunoreactive for the tachykinin substance P (SP). Few cells in the pars distalis immunoreact for SP. The SP-immunoreactive cells did not colocalize with beta-luteinizing hormone. RT-PCR confirmed the presence of preprotachykinin A mRNA in the PT. We hypothesize that SP, and possibly other preprotachykinin A-derived tachykinins, may play a role in the seasonal regulation of prolactin secretion in sheep.

The expression of a pre-B cell receptor (pre-BCR) is required for allelic exclusion and pre-BII cell differentiation. V(H)12 microH chains are unusual in that they form pre-BCRs and mediate allelic exclusion, but most cannot drive pre-BII cell differentiation. To explain this paradox, we examined pre-BCR functions and pre-BII cell differentiation in mice expressing microH chain transgenes encoding a B cell-permissible V(H)12 microH chain (designated 10/G4(6-1)), and a non-permissible V(H)12 microH chain (designated 8/G0). Compared with 10/G4 pre-BCRs, 8/G0 pre-BCRs are expressed at low levels on the cell surface. 8/G0 pre-BCRs mediate allelic exclusion, but 8/G0 pre-BII cells are defective in proliferation and expression of survival factors Bcl-2, Bcl-X(L) and hemokinin 1 (HK1). Increasing 8/G0 microH chain production restores HK1 transcription and improves proliferation of pre-BII cells as well as later stage B cell development. These data reveal a hierarchy of pre-BCR function that determines the development and plasticity of early B cells.

Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.

Background: For holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information., Results: Recently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques., Conclusion: In this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified.

Background: Tachykinins-like substance P (SP) have been shown to play an important role in initiating and perpetuating airway inflammation. Furthermore, they are supposed to be released into tissues in response to stress., Objective: The aim of this study was to investigate the effects of stress alone or in combination with allergic airway inflammation on SP expression in sensory neurons innervating the mouse airways., Methods: Balb/c mice were systemically sensitized to ovalbumin (OVA), followed by allergen aerosol exposure, and compared with non-sensitized controls. Additionally, OVA-sensitized and -challenged and non-sensitized mice were exposed to sound stress. SP expression in airway-specific and overall vagal sensory neurons of the jugular and nodose ganglion complex was analysed using retrograde neuronal tracing in combination with immunohistochemistry. Preprotachykinin A (PPT-A) mRNA, the precursor for SP, was quantified in lung tissue by real-time PCR. Bronchoalveolar lavage (BAL) fluid was obtained, and cell numbers and differentiation were determined., Results: Stress and/or allergic airway inflammation significantly increased SP expression in retrograde-labelled vagal sensory neurons from the mouse lower airways compared with controls [stress: 15.7+/-0.8% (% of retrograde-labelled neurons, mean+/-SEM); allergen: 17.9+/-0.4%; allergen/stress: 13.1+/-0.7% vs. controls: 6.3+/-0.3%]. Similarly, SP expression increased in overall vagal sensory neurons identified by the neuronal marker protein gene product (PGP) 9.5 [stress: 9.3+/-0.6% (% of PGP 9.5-positive neurons, means+/-SEM); allergen: 12.5+/-0.4%; allergen/stress: 10.2+/-0.4% vs. controls: 5.1+/-0.3%]. Furthermore, stress significantly increased PPT-A mRNA expression in lung tissue from OVA-sensitized and -challenged animals, and immune cells were identified as an additional source of SP in the lung by immunohistochemistry. Associated with enhanced neuronal SP expression, a significantly higher number of leucocytes were found in the BAL following allergen exposure. Further, stress significantly increased allergen-induced airway inflammation identified by increased leucocyte numbers in BAL fluids., Conclusion: The central event of sound stress leads to the stimulation of SP expression in airway-specific neurons. However, in sensitized stressed mice an additional local source of SP (probably inflammatory cells) might enhance allergic airway inflammation.

Hemokinin-1 is a recent addition to the family of mammalian tachykinins and is thought to play an important role in B cell and T cell lymphopoiesis. The mRNA coding for this peptide was expressed in some B lymphocyte cell lines including 70Z/3.12, ABE-8.1/2, and RAW8.1 cells, suggesting the possibility that hemokinin-1 may function in an autocrine or paracrine manner in these cells. Therefore, we quantified secretion of this peptide from the 70Z/3.12 cell line expressing hemokinin-1 mRNA. Despite a sensitive radioimmunoassay, we were surprised to find that hemokinin-1 secretion from confluent cells was below the level of detection of this assay. Furthermore, cell lysates routinely demonstrated a low or undetectable immunoreactive peptide. Collectively these studies show a limited production of hemokinin-1 peptide by transformed B cells.

The present study evaluated vasoactive intestinal peptide (VIP) and substance P (SP) mRNA expressions and the localization of both peptides in first- and third-trimester human placentas. VIP and SP mRNAs were detected by slot blot analysis in first- and third-trimester placental tissues. By immunohistochemistry both neuropeptides were localized in the trophoblast (syncytium and cytotrophoblastic cells) of the chorionic villi. Because little information is available on the role of VIP and/or SP on the secretion of placental hormones, we investigated the effect of these neuropeptides on human chorionic gonadotropin (hCG) and progesterone release from primary cultured human trophoblastic and JEG-3 cells. The addition of increasing doses of VIP resulted in a dose-dependent stimulation of hCG release from cultured human trophoblast and JEG-3 cells. Increasing doses of VIP also dose-dependently stimulated progesterone secretion from primary cultured trophoblastic cells at all time points evaluated and from JEG-3 cells only after 3 h. SP did not affect hCG and progesterone secretion either in cultured human trophoblast or in JEG-3 cells. In conclusion, the present study demonstrates that VIP and SP are mainly expressed in human trophoblasts, and that VIP modulates the in vitro secretion of hCG and progesterone, suggesting a different role in trophoblastic function of the two peptides.

We previously demonstrated that exogenously administered neurokinin A and neurokinin B, but not substance P, increased the sensitivity of cultured cerebellar granule neurons (CGNs) to glutamate. In the present study, the presence of tachykinin neuropeptides in CGNs was tested by confocal-based immunofluorescence. We found that neurokinin A and neurokinin B are present in CGNs but absent in astrocytes while substance P is abundant in astrocytes but absent in CGNs. It is postulated that the different localization of tachykinin neuropeptides in CGNs and astroglial cells has a physiological role in the modulation of excitatory transmission.

Background: The traditional neurotransmitter catecholamine and the neuropeptide tyrosine in sympathetic airway nerves have been proposed to be involved in the pathogenesis of airway diseases., Objective: The aim of the present study was to investigate the effect of allergic airway inflammation on the expression of catecholamine enzyme tyrosine hydroxylase (TH), neuropeptide tyrosine (NPY) and tachykinins in mouse sympathetic airway ganglia., Methods: Using neuronal tracing in combination with immunohistochemistry, the present study was designed to characterize TH, NPY and tachykinin profiles of superior cervical (SCG) and stellate ganglia after allergen challenge., Results: The vast majority of fast blue-labelled SCG neurons (allergen: 97.5+/-1.22% (mean+/-SEM) vs. controls: 94.5+/-1.48%, P=0.18) and stellate neurons (allergen: 95.3+/-1.01% vs. controls: 93.6+/-1.33%, P=0.34) were immunoreactive for TH. Of the TH immunoreactive and fast blue-labelled SCG neurons, 52.0+/-1.01% allergen vs. 51.2+/-3.58% controls (P=0.83) and stellate neurons, 57.3%+/-0.97 allergen vs. 56.4+/-1.65% controls (P=0.64) were positive for TH only but not NPY, whereas 45.3+/-1.05% allergen vs. 43.3+/-1.18% controls (P=0.47) of fast blue-labelled SCG neurons and 37.9+/-0.86% allergen vs. 37.1+/-1.24% controls (P=0.62) of fast blue-labelled stellate neurons were immunoreactive for both TH and NPY immunoreactivities. There was a trend of an increase, but not significant one, in the percentage of TH-/NPY-immunoreactive and fast blue-labelled neurons in allergen-treated animals in comparison with the controls. Tachykinins, however, were not expressed by sympathetic neurons and were also not induced in sympathetic neurons after allergen challenge., Conclusion: The present study indicates that allergic airway inflammation does not alter the expression of noradrenalin and NPY in sympathetic ganglia and also shows that sympathetic neurons do not respond to allergic airway inflammation with tachykinins induction. However, a participation of catecholamine and NPY in the pathogenesis of allergic airway inflammation cannot be excluded in the present study as a higher neurotransmitter output per neuron following allergen challenge could be possible.

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

In insects primary urine is produced by the Malpighian tubules under hormonal control. Here we have analysed the effects of the peptide locustatachykinin I (Lom-TK-I) on secretion in isolated Malphigian tubules. We also mapped the distribution of Lom-TK immunoreactivity in the gut in comparison with Locusta diuretic hormone (Lom-DH) and serotonin, two other factors that are active on locust tubules. Lom-TK-I produces an immediate, potent and long-lasting stimulation of fluid secretion. Furthermore, we show that Lom-TK-I acts synergistically with Lom-DH on fluid secretion and demonstrate that Lom-TKs are co-localised with Lom-DH in endocrine cells of the midgut ampullae. Thus, the two peptides might be released together to act synergistically on fluid secretion. Also serotonin and Lom-DH act synergistically and we can demonstrate a plexus of serotonin-containing axon processes over the midgut.

Projection neurons in the ventral striatum, the accumbens nucleus and olfactory tubercle, were examined by combining the retrograde tracing method and immunocytochemistry with antibodies against C-terminals of the preprodynorphin (PPD), preproenkephalin (PPE), preprotachykinin A (PPTA) and preprotachykinin B (PPTB). When the retrograde tracer was injected into the ventral pallidum, about 60% and 40% of retrogradely labeled neurons in the accumbens nucleus were immunoreactive for PPD and PPE, respectively. In contrast, all accumbens nucleus neurons projecting to the ventral mesencephalic regions including the substantia nigra and ventral tegmental area were immunopositive for PPD but not for PPE. Although no olfactory tubercle neurons projected fibers to the mesencephalic regions, 60% and 40% of olfactory tubercle neurons projecting to the ventrolateral portion of the ventral pallidum were immunoreactive for PPD and PPE, respectively, as were the accumbens nucleus neurons. About 70% of accumbens nucleus and olfactory tubercle neurons projecting to the ventral pallidum and all accumbens nucleus neurons projecting to the ventral mesencephalic regions showed PPTA immunoreactivity. A small population (2-12%) of accumbens neurons projecting to the ventral pallidum and mesencephalic regions displayed immunoreactivity for PPTB. Compared with the dorsal striatopallidal projection neurons that were reported to mostly express PPE, it was characteristic of the ventral striatum that only the smaller population (about 40%) of ventral striatopallidal projection neurons expressed PPE. This suggests that the ventral striatopallidal projection system is less specialized than the dorsal striatopallidal system in terms of peptide production, or that the ventral pallidum should be compared with a combined region of the globus pallidus and entopeduncular nucleus in the dorsal system.

Two peptides with limited structural similarity to mammalian substance P (SP) and neurokinin A (NKA) have been isolated from extracts of the intestine of the African clawed frog (Xenopus laevis). The primary structure of an SP-like peptide was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH(2), which is identical to the previously characterized peptide, bufokinin isolated from the toad Bufo marinus. The primary structure of an NKA-related peptide was established as Thr-Leu-Thr-Thr-Gly-Lys-Asp-Phe-Val-Gly-Leu-Met.NH(2). Only the five amino acids at the C-terminal region of the peptide are identical to mammalian NKA whereas the N-terminal region shows no structural similarity to previously characterized tachykinins. Immunohistochemical investigations of the gut wall revealed a dense network of nerve fibres and nerve cell bodies containing SP/NKA-like substances. The myotropic effects of the Xenopus tachykinins were compared with the contractile effect of mammalian SP and NKA on isolated strips of circular smooth muscle from Xenopus stomach. No significant differences in potencies (-log EC(50)) or in intrinsic activities were observed between the Xenopus and mammalian peptides. The potencies for the Xenopus SP-like (8.49+/-0.15) and the NKA-like peptide (8.12+/-0.06) were similar suggesting that the amino acid sequence at the N-terminal region of the tachykinins is not important in activating the tachykinin receptors in Xenopus gastric smooth muscle. The maximum response to Xenopus SP (alpha=0.59+/-0.06) was significantly lower than to the NKA-like peptide (alpha=1.0) suggesting a more effective interaction of the NKA-like peptide with the tachykinin receptor(s) in Xenopus stomach.

Preprodynorphin (PPD), preproenkephalin (PPE) and preprotachykinins A (PPTA) and B (PPTB) are known to be expressed by neostriatal projection neurons. In the present study, we investigated the distributions and colocalizations of immunoreactivities for those prepropeptides in the ventral striatum, such as the accumbens nucleus (Acb) and olfactory tubercle (OT). Antibodies raised against C-terminal portions of the prepropeptides labeled cell bodies of neurons with diameters of 8-15 microm. PPD-, PPE- and PPTA-immunoreactive neurons were distributed throughout the Acb and concentrated in the dense cell layer of the OT. PPTB-immunoreactive neurons were observed to form cell clusters, which were localized in mu-opioid receptor-immunoreactive patchy regions in the Acb, but were very rarely found in the dense cell layer of the OT. Double-immunofluorescence analysis revealed that PPD, PPE and PPTB immunoreactivities were shown in 69%, 19% and 14% of PPTA-immunoreactive neurons, respectively, in the Acb core region, and in 92%, 7% and 25% of PPTA-immunoreactive neurons, respectively, in the Acb shell region. In the olfactory bulb, 51%, 19% and 3% of PPTA-immunoreactive neurons showed PPD, PPE and PPTB immunoreactivities, respectively. PPD and PPE immunoreactivities were rarely coexpressed in single neurons of all striatal regions. The present results indicated that, although PPTA and PPE were occasionally coexpressed in single neurons of the ventral striatum, the segregated expression of PPD and PPE in the ventral striatum was similar to that in the dorsal striatum. The clustered localization of PPTB-expressing neurons in the Acb and near absence of PPTB-expressing neurons in the dense cell layer of the OT suggests that neurokinin B is a key substance in differentiating between the ventral and dorsal striatal regions., (Copyright 2002 IBRO)

Low doses of fenvalerate, a widely used type-II pyrethroid insecticide, have been shown previously to produce abnormal olfactory centers in the brain and abnormal olfactory-mediated behavior in beetles (Wegerhoff et al. [1998] Neuroreport 9:3241-3245). Here, we use the experimental advantages of the moth Manduca sexta to explore the cellular changes that lead to these abnormalities. Our results indicate that treatment with fenvalerate may affect multiple aspects of the development of the primary olfactory centers, the antennal lobes, in Manduca, including ingrowth of olfactory receptor axons, axon fasciculation, and targeting within the antennal lobe, and intercellular signaling between the receptor axons and the glial cells that ordinarily surround and stabilize the developing olfactory glomeruli., (Copyright 2001 Wiley-Liss, Inc.)

Tachykinin receptors NK1r, NK2r and NK3r bind tachykinins with different affinities and share pharmacological and molecular differences among animal species. NK1r, NK2r, NK3r and tachykinin (SP/NKA) distribution was studied by immunohistochemistry in the ileum of mouse since no data are available for this species. The results were then compared to those obtained in the rat and guinea pig either by us or by others to ascertain interspecies similarities and/or differences. NK1r- and NK3r-immunoreactivity (IR) were detected in neurons and NK1r-IR in the interstitial cells of Cajal at the deep muscular plexus. At variance with rat and guinea pig, NK1r-IR was also found in the myoid cells of the villi, while NK2r-IR was never detected in nerve varicosities. This latter datum suggests that the NK2r does not play a presynaptic role in the mouse. Unexpectedly, a high NK2r-IR and the presence of NK3r-IR were observed at the inner portion of the circular muscle layer in the mouse as well as in the rat and guinea pig, demonstrating a subregional distribution of these receptors. Tachykinin distribution did not show noticeable species-related differences. The present findings show species-related differences in the tachykinin receptor distribution that might be related to a different tachykinin control of intestinal motility.

Background: The study of periodontitis provides a unique model for assessing the involvement of neuropeptides in inflammatory disease., Aim: To investigate the effects of periodontal treatment, resulting in a return to periodontal health, on the levels of substance P (SP) and neurokinin A (NKA) in gingival crevicular fluid (GCF)., Method: We completed a cause of non-surgical treatment for 8 subjects with periodontitis (6 females 2 males, mean age 45.1, range 38-67 years) started a course of non-surgical periodontal treatment. Clinical indices were measured at 2 periodontitis sites at the initial visit and at 8 weeks after the completion of treatment in each subject. A 30-s sample of GCF was collected from each test site using perio paper strips. Each strip was placed into 500 microl of ice cold 0.1 M PBS, pH 7.4, vortex mixed for 30 s, and then stored at -70 degrees C until analysed by radioimmunoassay., Results: The clinical condition of all test sites improved as a result of the periodontal treatment. The levels (pg/30 s sample) of SP fell from 56.3 (SD 66.0) at the initial visit to 4.2 (3.1) after treatment, p=0.017. The concentration (pg/microl) of SP in GCF fell from 140.6 (175.6) to 24.2 (11.1), p=0.036. The levels of NKA fell from 30.5 (17.1) to 10.6 (4.9), p=0.012 whereas the concentration changed little from 85.4 (43.5) to 61.6 (15.1), p=0.41., Conclusion: The reduction in inflammation resulting from effective periodontal treatment is associated with a reduction in the levels of tachykinins in gingival crevicular fluid.

The localization, release, and effects of substance P and neurokinin A were studied in the porcine pancreas and the localization of substance P immunoreactive nerve fibers was examined by immunohistochemistry. The effects of electrical vagus stimulation and capsaicin infusion on tachykinin release and the effects of substance P and neurokinin A infusion on insulin, glucagon, somatostatin, and exocrine secretion were studied using the isolated perfused porcine pancreas with intact vagal innervation. NK-1 and NK-2 receptor antagonists were used to investigate receptor involvement. Substance P immunoreactive nerve fibers were localized to islets of Langerhans, acini, ducts, and blood vessels. Vagus stimulation had no effect on substance P and neurokinin A release, whereas capsaicin infusion stimulated release of both. Substance P and neurokinin A infusion increased release of insulin, glucagon, and exocrine secretion, whereas somatostatin secretion was unaffected. The effect of substance P on insulin, glucagon, and exocrine secretion was blocked by the NK-1 receptor antagonist. The effect of electrical stimulation of vagus nerves on insulin and exocrine secretion was not influenced by tachykinin receptor antagonists. We conclude that tachykinins stimulate both endocrine and exocrine pancreatic functions through NK-1 receptors. Tachykinins are not involved in vagal regulation of pancreatic secretion in pigs but could constitute part of an alternative stimulatory system.

Off-axis illumination elicits lateral inhibition at the primary visual synapse in crustacea and insects. The evidence suggests that the inhibitory action is presynaptic (i.e., on the photoreceptor terminal) and that the amacrine neurons of the lamina ganglionaris (the first synaptic layer) may be part of the inhibitory pathway. The neurotransmitters and the synaptic mechanisms are unknown. We show by immunocytochemistry that GABA and a tachykinin-related peptide (TRP) are localized in the amacrine neurons of the crayfish lamina ganglionaris. Indirect evidence suggests that GABA and TRP may be colocalized in these neurons. The extensive processes of the amacrine neurons occupy lamina layers containing the terminals of photoreceptors. Application of exogenous GABA and TRP to photoreceptor terminals produces a short-latency, dose-dependent hyperpolarization with a decay time constant on the order of a few seconds. TRP also exhibits actions that evolve over several minutes. These include a reduction of the receptor potential (and the light-elicited current) by approximately 40% and potentiation of the action of GABA by approximately 100%. The mechanisms of TRP action in crayfish are not known, but a plausible pathway is a TRP-dependent elevation of intracellular Ca(2+) that reduces photoreceptor sensitivity in arthropods. Although the mechanisms are not established, the results indicate that in crayfish photoreceptors TRP displays actions on two time scales and can exert profound modulatory control over cell function.

Sixty days following neonatal dopamine depletion (>98%) with 6-hydroxydopamine, preprotachykinin and preprodynorphin mRNA levels were significantly reduced (67 and 78% of vehicle controls, respectively) in the anterior striatum as determined by in situ hybridization while preproenkephalin mRNA expression was elevated (133% of vehicle controls). Suppression of the serotonin hyperinnervation phenomenon in the dopamine-depleted rat with 5,7-dihydroxytryptamine yielded no significant alterations in reduced striatal preprotachykinin (66%) or preprodynorphin (64%) mRNA levels, while preproenkephalin mRNA expression remained significantly elevated (140%). These data suggest that striatal serotonin hyperinnervation does not contribute to the development of dysregulated striatal neuropeptide transmission in either direct or indirect striatal output pathways following neonatal dopamine depletion.

In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neurons had been achieved by incubating the tissue with capsaicin (10 microM) for 15 min. At immunohistochemistry, tachykinin- and CGRP-immunoreactivities (TK-IR/CGRP-IR) were detected in varicose nerve fibers throughout the common bile duct, while TK-IR cell bodies were observed in the terminal portion (ampulla) only. In vivo pretreatment with capsaicin (50 mg/kg; 6-7 days before) decreased the number of CGRP-IR nerves, whereas the TK-IR neural network was apparently unchanged. In conclusion, our data provide functional evidence for the presence of capsaicin-sensitive primary afferent nerve endings in the guinea-pig terminal biliary tract, whose stimulation by capsaicin or EFS produces the release of tachykinins and CGRP. In addition, morphological evidence is provided that the bulk of TK-IR material in the biliary tract is contained in intrinsic neuronal elements, while CGRP in this tissue is of extrinsic origin only. Tachykinins, probably released in small amounts by capsaicin, act by activating receptors of the NK1, NK2 and NK3 type, most probably located on intrinsic cholinergic neurons, which in turn release ACh to produce the final excitatory motor response. The contractile response to capsaicin obtained in the presence of the three tachykinin receptor antagonists could be due to the co-released CGRP and/or to other unknown neurotransmitters. CGRP produces either indirect excitatory or direct inhibitory responses by stimulation of CGRP2 and CGRP1 receptors, respectively.

The activity of the adult stomatogastric ganglion (STG) depends on a large number of aminergic and peptidergic modulatory inputs. Our aim is to understand the role of these modulatory inputs in the development of the central pattern-generating networks of the STG. Therefore, we analyze the developmental and adult expressions of three neuropeptides in the stomatogastric nervous system of the lobsters Homarus americanus and Homarus gammarus by using wholemount immunocytochemistry and confocal microscopy. In adults, red pigment-concentrating hormone (RPCH)-like, proctolin-like, and a tachykinin-like immunoreactivity are present in axonal projections to the STG. At 50% of embryonic development (E50), all three peptides stain the commissural ganglia and brain, but only RPCH- and proctolin-like immunoreactivities stain axonal arbors in the STG. Tachykinin-like immunoreactivity is not apparent in the STG until larval stage II (LII). The RPCH-immunoreactive projection to the STG consists of two pairs of fibers. One pair stains for RPCH immunoreactivity at E50; the second RPCH-immunoreactive pair does not stain until about LII. One pair of the RPCH fibers double labels for tachykinin-like immunoreactivity. The adult complement of neuromodulatory inputs is not fully expressed until close to the developmental time at which major changes in the STG motor patterns occur, suggesting that neuromodulators play a role in the tuning of the central pattern generators during development.

Because cholecystokinin (CCK) acts as a "functional" endogenous opioid antagonist, it has been proposed that changes in central CCKergic neurotransmission might account for the relative resistance of neuropathic pain to the analgesic action of morphine. This hypothesis was addressed by measuring CCK-related parameters 2 weeks after unilateral sciatic nerve section in rats. As expected, significant decreases (-25-38%) in the tissue concentrations and in vitro release of both substance P and calcitonin gene-related peptide were noted in the dorsal quadrant of the lumbar spinal cord on the lesioned side. In contrast, the tissue levels and in vitro release of CCK were unchanged in the same area in lesioned rats. Measurements in dorsal root ganglia at L4-L6 levels revealed no significant changes in proCCK mRNA after the lesion. However, sciatic nerve section was associated with a marked ipsilateral increase in both CCK-B receptor mRNA levels in these ganglia (+70%) and the autoradiographic labeling of CCK-B receptors by [3H]pBC 264 (+160%) in the superficial layers of the lumbar dorsal horn. Up-regulation of CCK-B receptors rather than CCK synthesis and release probably contributes to increased spinal CCKergic neurotransmission in neuropathic pain.

Sensory afferents in the thoracic ganglia of the locust Locusta migratoria were labelled with antisera to different neuropeptides: locustatachykinins, FMRFamide and allatotropin. The locustatachykinin-immunoreactive (LTKIR) sensory fibres were derived from the legs and entered the ventral sensory neuropil of each of the thoracic ganglia via nerve 5. In the thoracic neuropil, the LTKIR sensory fibres formed a distinct plexus of terminations ventrally in the ipsilateral hemisphere. The peripheral cell bodies of the sensory neurones could not be revealed, but lesion experiments indicated that origin of the LTKIR fibres was the tarsus of each leg. Possibly the thin fibres are from tarsal chemoreceptors. Double labelling immunocytochemistry revealed that all the LTKIR sensory fibres contained colocalized FMRFamide immunoreactivity. A larger population of sensory fibres reacted with antiserum to moth (Manduca sexta) allatotropin. By means of double labelling immunocytochemistry, we could show that the LTKIR fibres constituted a subpopulation of the larger set of allatotropin-like immunoreactive fibres. Thus some sensory fibres may contain colocalized peptides related to locustatachykinins, FMRFamide-related peptide(s) and allatotropin-like peptide. A separate non-overlapping small set of sensory fibres in nerve 5 reacted with an antiserum to serotonin. Sensory fibres of the other nerves of the ventral nerve cord, including the abdominal ganglia, did not react with the peptide antisera. Since acetylcholine is the likely primary neurotransmitter of insect sensory fibres, it is possible that the peptides and serotonin are colocalized with this transmitter and serve modulatory functions in a subset of the leg afferents., (Copyright 1999 Elsevier Science B.V.)

Peptides with sequence similarities to members of the tachykinin family have been identified in a number of invertebrates belonging to the mollusca, echiuridea, insecta and crustacea. These peptides have been designated tachykinin-related peptides (TRPs) and are characterized by the preserved C-terminal pentapeptide FX1GX2Ramide (X1 and X2 are variable residues). All invertebrate TRPs are myostimulatory on insect hindgut muscle, but also have a variety of additional actions: they can induce contractions in cockroach foregut and oviduct and in moth heart muscle, trigger a motor rhythm in the crab stomatogastric ganglion, depolarize or hyperpolarize identified interneurons of locust and the snail Helix and induce release of adipokinetic hormone from the locust corpora cardiaca. Two putative TRP receptors have been cloned from Drosophila; both are G-protein coupled and expressed in the nervous system. The invertebrate TRPs are distributed in interneurons of the CNS of Limulus, crustaceans and insects. In the latter two groups TRPs are also present in the stomatogastric nervous system and in insects endocrine cells of the midgut display TRP-immunoreactivity. In arthropods the distribution of TRPs in neuronal processes of the brain displays similar patterns. Also in coelenterates, flatworms and molluscs TRPs have been demonstrated in neurons. The activity of different TRPs has been explored in several assays and it appears that an amidated C-terminal hexapeptide (or longer) is required for bioactivity. In many invertebrate assays the first generation substance P antagonist spantide I is a potent antagonist of invertebrate TRPs and substance P. Locustatachykinins stimulate adenylate cyclase in locust interneurons and glandular cells of the corpora cardiaca, but in other tissues the putative second messenger systems have not yet been identified. The heterologously expressed Drosophila TRP receptors coupled to the phospholipase C pathway and could induce elevations of inositol triphosphate. The structures, distributions and actions of TRPs in various invertebrates are compared and it is concluded that the TRPs are multifunctional peptides with targets both in the central and peripheral nervous system and other tissues, similar to vertebrate tachykinins. Invertebrate TRPs may also be involved in developmental processes.

The midgut of the African migratory locust, Locusta migratoria, was found to contain endocrine-like cells that stained positively for locustatachykinin I (Lom TK I)-like immunoreactivity. These cells were distributed in an unequal manner throughout the midgut of the locust, with a greater density of Lom TK I-like immunoreactive endocrine-like cells occurring in the posterior region of the midgut. These singly occurring cells appear elongate with an apical extension projecting toward the midgut lumen and a smaller projection extending towards the midgut basal lamina. No immunoreactive neuronal processes were detected along the midgut wall. Radioimmunoassays revealed that the female midgut contained two to three times more Lom TK I-like material than the male midgut, and radioimmunoassay coupled to high-performance liquid chromatography analysis revealed that at least five locustatachykinin isoforms appear to be present in the midgut. This distribution of Lom TK I-like material suggests possible functional differences in the various regions of the midgut. The role that these cells may play in locust midgut secretory activity and motility remains unknown. However, the addition of synthetic Lom TK I through IV to a ring type midgut muscle preparation stimulated contraction of midgut circular muscles, suggesting a possible physiological role for these peptides. Dose-response curves constructed for Lom TK I-IV revealed that the peptide-induced contractions increased in a dose-dependent manner.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor with a therapeutic potential in neurodegenerative disorders. GDNF is expressed in the adult striatum, but its signalling tyrosine kinase receptor, c-ret, has not been detected in this structure by in situ hybridization. In the present work, we first examined c-ret and GDNF receptor alpha 1 (GFR-alpha 1) expression using an RNAse protection assay, and found that both receptors are expressed in the adult rat striatum. We then examined whether GDNF was able to regulate the phenotype and/or prevent the degeneration of striatal projection neurons in a well-characterized model of excitotoxic damage. A fibroblast cell line, engineered to overexpress GDNF, was grafted in adult rats striatum 24 h before quinolinic acid (QUIN) injection. QUIN injection alone or in combination with the control cell line induced a loss of glutamic acid decarboxylase 67 (GAD)-, preprotachykinin A (PPTA)-, prodynorphin (DYN)- and preproenkephalin (PPE)-positive neurons. GDNF selectively prevented: (i) the loss of a subpopulation of striatonigral neurons expressing GAD and PPTA; (ii) the atrophy of PPTA-positive neurons; and (iii) the decrease in GAD, PPTA and DYN mRNA expression, after QUIN injection. Moreover, in unlesioned animals, GDNF increased the size of PPTA-positive neurons and up-regulated their mRNA levels. In contrast, GDNF showed no effect in intact or lesioned striatopallidal PPE-positive neurons. Thus, our findings show that GDNF selectively regulates the phenotype and protects striatonigral neurons from QUIN-induced excitotoxicity, suggesting that GDNF may be used for the treatment of striatonigral degenerative disorders, e.g. Huntington's disease and multiple system atrophy.

Neurons expressing preprotachykinin A and preprotachykinin B, which are the precursor prepropeptides of substance P and neurokinin B (neuromedin K), respectively, were characterized immunocytochemically in the rat neocortex. Antibodies raised against C-terminal portions of preprotachykinins were used for labeling cell bodies of preprotachykinin-producing neurons. Neurons immunoreactive for preprotachykinin B were encountered four times more frequently in the neocortex than those immunoreactive for preprotachykinin A. Preprotachykinin A-immunoreactive neurons were scattered more frequently in the deep cortical layers (layers IV-VI) than in the superficial layers (layers I-III), whereas preprotachykinin B-immunoreactive neurons were distributed more frequently in the superficial layers than in the deep layers. Almost all preprotachykinin-expressing neurons were immunoreactive for GABA, suggesting that they were non-pyramidal cells. However, co-expression of the two preprotachykinin immunoreactivities in single neurons was not found. Preprotachykinin-expressing neocortical neurons were further examined with markers for subpopulations of GABAergic cortical neurons. Immunoreactivities for parvalbumin, calbindin and somatostatin were found in 69%, 27% and 11%, respectively, of preprotachykinin A-immunoreactive neurons. Conversely, preprotachykinin A-immunoreactive neurons constituted only 6% of parvalbumin-immunoreactive neurons, 4% of calbindin-immunoreactive neurons and 1% of somatostatin-immunoreactive neurons. Immunoreactivities for calretinin, choline acetyltransferase, vasoactive intestinal polypeptide, corticotropin-releasing factor and cholecystokinin were detected in 13-39% of preprotachykinin B-immunoreactive neurons. Preprotachykinin B immunoreactivity was seen in 33% of calretinin-positive neurons, 45% of cholinergic neurons, 47% of vasoactive intestinal polypeptide-positive neurons, 59% of corticotropin-releasing factor-positive neurons and 83% of cholecystokinin-positive neurons. These results indicate that preprotachykinin A- and preprotachykinin B-expressing neurons constitute separate populations of GABAergic non-pyramidal neurons in the neocortex. Since receptors for substance P and neurokinin B are expressed in GABAergic neurons [Kaneko T. et al. (1994) Neuroscience 60, 199-211] and pyramidal neurons [Ding Y. Q. et al. (1996) J. comp. Neurol. 364, 290-310], respectively, cortical neurons may use two separate lines of tachykinin signals; substance P serves as a signal between GABAergic non-pyramidal neurons, whereas neurokinin B acts as a signal of GABAergic neurons to pyramidal neurons.

Toluene diisocyanate (TDI) is a potent sensitizer that causes occupational asthma in a significant proportion of subjects exposed. We used an animal model to investigate whether neuropeptide changes occur in the airways of immunized and TDI-challenged guinea pigs. Animals were immunized by weekly intradermal injections, challenged with TDI (5 to 20 ppb) after the third injection, and killed 6 h after exposure. Control guinea pigs received injections of saline. Lung tissue was processed immediately and analyzed for nerves using the streptavidin-biotin complex peroxidase method with antisera to the neural marker protein gene product 9.5 (PGP 9.5), substance P (SP), and calcitonin gene- related peptide (CGRP). We also quantified the inflammatory infiltrate in the submucosa of central airways, and we measured the serum level of specific IgG and IgG1. Specific antibodies against TDI were present only in immunized animals. Immunized as compared with nonimmunized animals had a significant increase in eosinophils in the submucosa of central airways, and a further increase was observed 6 h after TDI challenge. Immunization and TDI challenge did not modify the number of mononuclear cells in the submucosa of central airways in both nonimmunized and immunized animals. TDI exposure did not change the overall innervation in both nonimmunized and immunized animals, but the density of PGP 9.5-positive nerves was significantly different between nonimmunized and immunized TDI-challenged animals. The density of SP-, and CGRP-immunostained nerves was significantly lower in immunized TDI-challenged than in nonimmunized animals. TDI exposure significantly decreased the density of SP-positive nerves in nonimmunized animals. A negative relationship was found between the presence of airway inflammation, as indexed by eosinophil cell infiltration, and the density of PGP 9.5-, SP-, and CGRP-immunostained nerves. In conclusion, TDI produces airway inflammation and neuropeptides changes in the central airways of immunized guinea pigs 6 h after TDI challenge. These findings support an interaction between tachykinins, inflammatory (i.e., eosinophils) and possibly immune cells.

The native tachykinins cod neurokinin A and cod substance P, serotonin and acetylcholine have excitatory effects on the circular smooth muscle of the cod intestine. Furthermore, immunoreactivities to the cod tachykinins, serotonin and two markers for cholinergic neurones, viz. choline acetyltransferase and vesicular acetylcholine transporter, have been demonstrated in myenteric neurones of the cod intestine. In order to elucidate whether the neurones containing these substances project orally and thus might be involved in the ascending excitatory reflex of peristalsis, myotomy operations have been performed on the cod intestine. The immunoreactive areas of the myenteric plexus immediately oral and anal to the myotomy operations have been measured by using confocal laser scanning microscopy. Large accumulations of immunoreactivity to the tachykinins are found on the anal side of the myotomies, indicating oral projections of tachykininergic neurones. The areas immunoreactive to serotonin and choline acetyltransferase are of equal size on the oral and anal sides. Since the tachykinin containing neurones of the intestine project orally, and since cod neurokinin A and cod substance P have excitatory effects on circular smooth muscle, we conclude that tachykininergic neurones are involved in the ascending excitatory reflex of peristalsis in the cod intestine.

The central complex, a highly ordered neuropil area in the insect brain, plays a role in motor control and spatial orientation. To further elucidate the neurochemical architecture of this brain area, we have investigated the distribution and morphology of neurons containing locustatachykinin I/II-related substances in the central complex of the locust Schistocerca gregaria. The central complex is innervated by at least 66 locustatachykinin I/II-immunoreactive neurons, which belong to two sets of tangential neurons and four sets of columnar neurons. These neurons give rise to immunostaining in the protocerebral bridge, in several layers of the upper division of the central body, and in all layers except layer 5 of the lower division of the central body. Double-label experiments show colocalization of immunoreactivity for both locustatachykinin I/II and octopamine in tangential neurons of the protocerebral bridge. A pair of tangential neurons of the lower division of the central body exhibits both locustatachykinin I/II and gamma-aminobutyric acid (GABA) immunoreactivity. A set of 16 columnar neurons of the lower division of the central body shows colocalized immunoreactivity for locustatachykinin II, leucokinin, and substance P. This study reveals novel features of the anatomical organization of the locust central complex and suggests a prominent role for locustatachykinin-related peptides as neuromediators and cotransmitters within this brain area.

Tachykinins form a family of peptides with neurotransmitter/neuromodulator function. Four tachykinins, substance P, neurokinin A, neuropeptide gamma and neuropeptide K, are encoded by the same PreProTachykinin (PPT) gene. Alternatively spliced mRNAs encode different combinations of these peptides (Brown, E.R., Harlan, R.E., Krause, J.E., Gonadal steroid regulation of substance P (SP) and SP-encoding mRNA in the rat anterior pituitary and hypothalamus, Endocrinology, 126 (1990) 330-340; Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S., Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. USA, 84 (1987) 881-885). The proportion of PPT mRNAs varies from tissue to tissue (Carter, M.S., Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma, J. Neurosci., 10 (1990) 2203-2214), and within the rat hypothalamus according to the estrous cycle-related hormonal status (Gautreau, A., Duval, P., Kerdelhué, B., Variations in substance P-encoding preprotachykinin and substance P receptor NK-1 mRNA transcripts in the rat hypothalamus throughout the estrous cycle: a correlation between amounts of beta-preprotachykinin and NK-1 mRNA, Mol. Brain Res., (1997) in press). Tachykinin receptors as well as tachykinins are regulated at the mRNA level. A fully quantitative method is needed to deal with the complex physiological regulation of the tachykinin system. Here, we describe an RNase protection assay that allows the simultaneous quantitation of alternatively spliced PPT mRNAs, Substance P receptor NK-1 mRNA, and glyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA as an internal control, in the rat hypothalamus. The advantages of this method are its high sensitivity (0.1 pg) and a wide range of linearity (more than 3 orders of magnitude). Moreover, this protocol provides guidelines to set up a quantitative multiprobe RNase protection assay for other genes.

Abnormalities of the enteric nervous system are thought to explain the pathophysiology of motility disorders. Our aim was to determine if particular classes of enteric neurons are affected in slow transit constipation (STC). Specimens were taken from the terminal ileum and ascending, transverse and descending colon of patients undergoing subtotal colectomy for STC. Immunohistochemistry was performed using antisera to neuron-specific enolase, tachykinin, leu-enkephalin, choline acetyltransferase, vasoactive intestinal peptide, nitric oxide synthase, tyrosine hydroxylase and neuropeptide Y. The density of nerve fibres labelled with these antibodies in each layer was compared with age-matched controls. The density of nerve fibres with tachykinin and enkephalin immunoreactivity was reduced in the colonic circular muscle of the 15 patients with STC, whereas innervation of all other layers was normal. This reduction of tachykinin-immunoreactive nerve fibres also occurred in nine of the 12 specimens of terminal ileum examined. No difference was detected in the density or distribution of nerve fibres using the other antisera. Excitatory nerve fibres are present in the circular muscle in STC but they are deficient in tachykinins and enkephalin.

Four tachykinin-related peptides, locustatachykinin 1-4 (LomTK 1-4) are distributed in interneurons throughout the central nervous system of the locust Locusta migratoria and may have important roles as neurotransmitters or neuromodulators. In search of the central actions of LomTKs, we analyzed the response of the efferent dorsal unpaired median (DUM) neurons in the locust metathoracic ganglion. Immunocytochemistry, using an antiserum against LomTK 1, combined with intracellular filling of efferent DUM neurons with Lucifer yellow, revealed that LomTK-immunoreactive fibers are in close proximity to dendritic arborizations of the DUM neurons. Hence, LomTKs may act on DUM neurons by releasing locally in the metathoracic ganglion. Intracellular recordings were made from somata of DUM neurons, and LomTKs were either bath-applied to an isolated metathoracic ganglion or pressure-ejected onto the DUM neuron soma. LomTK 1 at concentrations of 0.1 mM-0.1 microM caused a relatively slow, reversible depolarization with a subsequent increase in the frequency of action potential firing. Amino-terminally truncated forms of LomTK 1 were applied to DUM neurons. The heptapeptide [3-9]-LomTK 1 had a substantially reduced activity, and bioactivity was lost after further truncation. Spantide 1, an antagonist of mammalian tachykinin receptors, reversibly blocked the effect of LomTK 1. The effect of LomTK 1 was clearly reduced in the presence of GDP-beta-S, a stable analog of GDP that inactivates G-proteins. The action of LomTK 1 was potentiated by both IBMX and theophylline, two cyclic AMP (cAMP) phosphodiesterase inhibitors. The action of LomTK 1 was mimicked by pressure-ejecting 8-bromo-cAMP, a membrane permeable analog of cAMP, and by forskolin, an adenylate cyclase activator. Furthermore, cAMPS, a blocker of protein kinase A activity, reduced the effect of LomTK 1. These findings indicate that cAMP is involved in mediating DUM neuron depolarization.

Background & Aims: Functional classes of enteric neurons in small mammals can be determined by neurochemical coding and axonal projections. The aim of this study was to describe the projections of vasoactive intestinal peptide (VIP)-, tachykinin (TK)-, and calretinin-immunoreactive (IR) interneurons and circular muscle motor neurons in human colon., Methods: Human colonic myenteric neurons were retrogradely filled with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (DII) from either the myenteric plexus or the circular muscle layers in organotypic culture preparations. The preparations were then labeled with antisera, and the position and immunoreactivity of DII-filled neurons was recorded., Results: Nine percent of circular muscle motor neurons were labeled with TK immunoreactivity and these projected orally; 22% were VIP-IR and projected anally. Fifty-one percent of myenteric neurons that projected anally to the myenteric ganglia were VIP-IR and none were TK-IR. In contrast, 23% of neurons projecting orally were TK-IR but only 2% were VIP-IR. Calretinin immunoreactivity was present in 23% of neurons projecting anally and 3% projecting orally within the myenteric plexus. Six percent of circular muscle motor neurons had Dogiel type II morphology, and 50% of these were TK-IR., Conclusions: Circular muscle motor neurons and myenteric interneurons with TK, VIP, and calretinin immunoreactivity in the human colon have distinct projections and polarity.

The distribution of nerves containing calcitonin gene-related peptide (CGRP), substance P (SP), neurokinin A (NKA), and vasoactive intestinal peptide (VIP) was examined in the human urinary bladder, using both single- and double-label immunohistochemistry. Nerves containing CGRP and tachykinins were typically present within the subepithelial region, encircling intramural ganglia and around blood vessels. These nerves were sparsely distributed, and only very rarely projected to the smooth muscle bundles of the detrusor. In contrast, VIP-containing nerves formed a dense subepithelial plexus and also projected to the detrusor muscle bundles. The double-label studies revealed that approximately 26% +/- 10% of CGRP-immunoreactive nerves also contained SP and NKA, but that no CGRP fibers coexpressed VIP. Conversely, all SP-immunoreactive fibers also contained CGRP, and many coexpressed NKA. At least three neurochemically distinct populations of nerves can therefore be discerned in the human bladder: VIP/-, CGRP/-, and CGRP/SP/ +/- NKA. The density of CGRP and SP-immunoreactive nerves within the subepithelium of 14 women with urodynamically-proven idiopathic detrusor instability was increased by 82% (p = 0.003) and 94% (p = 0.036), respectively, relative to that of 14 control women with no symptoms of frequency or urgency. This effect was not due to an increase in overall nerve density, because immunoreactivity for the general nerve marker, protein gene product, was not significantly different between the two groups (p = 0.21). The results indicate that at least some patients with detrusor instability demonstrate increases in a specific class of nerve fibers containing CGRP and SP. These peptides are characteristic of sensory neurons in a number of species, suggesting that abnormalities in the afferent arm of the micturition reflex may be associated with detrusor instability.