Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs

Calcitonin gene-related peptide (CGRP) has been immunohistochemically co-localized with substance P (SP) in capsaicin-sensitive, varicose axons supplying the skin, viscera and cardiovascular system of the guinea pig. After treatment with colchicine in vitro, 82% of SP neurons in the dorsal root ganglia contained CGRP-like immunoreactivity while 96% of CGRP neurons were immunoreactive for SP. Both CGRP- and SP-like immunoreactive material are transported peripherally and centrally from dorsal root ganglia. Thus, in tissues such as the gut where there are intrinsic nerves containing SP but lacking CGRP, CGRP-like immunoreactivity is a useful means of specifically labelling axons of most sensory neurons containing SP.     

Histamine application to the nasal mucosa induces release of calcitonin gene-related peptide and substance P from peripheral terminals of trigeminal ganglion: a morphological study in the guinea pig.

Short-term effects of application of histamine to the nasal mucosa on trigeminal ganglion neurons containing calcitonin gene-related peptide (CGRP) and substance P (SP) were examined in guinea pig. Immunoreactivities to CGRP and SP in these neurons were decreased 30 min after the histamine application. The decreases were most marked at 1-3 h after application, after which the immunoreactivities began to increase, reaching the base line by 6 h after the application. The immunoreactivities to CGRP and SP in the nerve endings of nasal mucosa were not decreased. The expression of mRNAs for both peptides in the soma of trigeminal neurons was unchanged. The histamine application to the nasal mucosa may cause release of CGRP and SP from terminals of peripheral processes of trigeminal ganglion neurons, and enhance axonal transport of these peptides, but does not affect their biosynthesis in the soma of trigeminal ganglion neurons.     

Simultaneous release of substance P- and calcitonin gene-related peptide (CGRP)-like immunoreactivity from isolated muscle of the guinea pig urinary bladder

Capsaicin (10 microM) induced a tetrodotoxin (TTX)-resistant release of substance P (SP)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) from muscle strips of the guinea pig isolated urinary bladder. A second application of capsaicin had no effect, indicating a specific effect on sensory nerves (desensitization). In functional experiments, capsaicin produced a phasic contraction of isolated bladder strips. This response was TTX-resistant, exhibited desensitization and was specifically antagonized by [D-Pro4, D-Trp7.9, Phe11] SP(4-11) a SP antagonist which also reduced, at a similar extent, the contraction induced by exogenous SP. These findings provide direct neurochemical and functional evidence for a transmitter role for a SP-like peptide(s) from peripheral sensory terminals in the guinea pig urinary bladder.     

Coexistence of vasoactive intestinal polypeptide- and substance P-like immunoreactivities in the tongue of the guinea pig

Vasoactive intestinal polypeptide (VIP)- and substance P (SP)-like immunoreactivities were examined in the tongue of the guinea pig by using the double immunofluorescence method. Coexistence of VIP- and SP-like immunoreactivities was suggested in many nerve fibers innervating the lingual salivary glands, as well as in a few intralingual ganglionic cells.     

Immunocytochemical analysis of potential neurotransmitters present in the myenteric plexus and muscular layers of the corpus of the guinea pig stomach

Recent electrophysiological studies of neurons of the myenteric plexus of the corpus of the guinea pig stomach have revealed that slow synaptic events are extremely rare. In contrast, they are commonly encountered in similar investigations of myenteric ganglia of the guinea pig small intestine. The current immunocytochemical analysis of the myenteric plexus and innervation of the muscularis externa of the corpus of the guinea pig stomach was undertaken in order to determine whether putative neurotransmitters capable of mediating slow synaptic events are present in gastric ganglia. A major difference between the small intestine and the stomach was found in the innervation of the musculature. Whereas the longitudinal muscle layer of the small intestine contains very few nerve fibers and is innervated mainly at its interface with the myenteric plexus, the longitudinal muscle of the corpus of the stomach contained as many varicose substance P (SP)-, vasocative intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive axons as the circular muscle layer. These putative neurotransmitters were also present in the ganglia of the myenteric plexus, where varicose SP-, VIP-, and NPY-immunoreactive fibers encircled nonimmunoreactive neurons. Varicose 5-hydroxytryptamine (5-HT)-immunoreactive terminal axons were essentially limited to the myenteric plexus and were found both in ganglia and in interganglionic connectives, where they were particularly numerous; 5-HT-immunoreactive neurons appeared to be more abundant in the stomach than in the small intestine. Tyrosine hydroxylase (TH)- and calcitonin-gene-related-peptide (CGRP)-immunoreactive axons were also more common in the myenteric plexus than in the musculature, but of these, only the TH-immunoreactive neurites tended, like those of the other putative transmitters, to encircle neurons in myenteric ganglia. Evidence was obtained that, as in the small intestine, at least some of the SP-, VIP-, NPY-, and 5-HT-immunoreactive fibers in the stomach are derived from intrinsic gastric myenteric neurons. In contrast, unlike the small intestine, gastric myenteric ganglia appeared to lack intrinsic CGRP-immunoreactive neurons; therefore, the CGRP-immunoreactive gastric axons are probably of extrinsic origin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical Properties of the Peripheral Neurons Projecting to the Pig Bulbospongiosus Muscle

Retrograde neuronal tracing and single labelling immunofluorescence methods were used to define the neurochemical content of the peripheral autonomic and sensitive neurons projecting to the male pig striated bulbospongiosus muscle (BSM). The retrograde fluorescent neuronal tracer Fast Blue (FB) was injected into the left bulbospongiosus muscle of four intact impuberal pigs. After a 10‐day survival time, the ipsilateral sacral sympathetic trunk ganglia (STGs), the caudal mesenteric ganglion (CMG), and the sacral spinal ganglia (SGs) were collected from each animal. In FB+ neurons of these ganglia, the presence of cathecolamine‐ (tyrosine hydroxylase‐TH), acetylcholine‐ (vesicular acetylcholine transporter‐VChAT), or nitric oxide‐synthesizing (neuronal Nitric Oxide Synthase‐nNOS) enzymes and of some biologically active peptides (calcitonine gene‐related peptide‐CGRP, Leu‐Enkephaline‐LENK, Neuropeptide Y‐NPY, Substance P‐SP and Vasoactive Intestinal Peptide‐VIP) were studied. The ipsilateral STGs FB+ neurons showed immunoreactivity principally for TH and NPY and in decreasing order for VIP, VChAT, SP, CGRP, nNOS, and LENK. The left CMG FB+ neurons were immunoreactive to TH and NPY, and in smaller proportions for VIP, LENK, VChAT, CGRP, nNOS, and SP. The ipsilateral SGs FB+ neurons resulted immunoractive for CGRP, LENK, NPY, nNOS, SP, and VChAT. The heterogeneous neurochemical content of the peripheral neurons projecting to the pig BSM allows us to hypothesize the involvement of autonomic ganglia in the activity of both blood vessels and striated fibers of the muscle and the involvement of sensory ganglia in the afferent transmission from the muscle to spinal cord and in antidromic mechanisms that causes the relaxation of the BSM blood vessels. Anat Rec, 299:1192–1202, 2016. © 2016 Wiley Periodicals, Inc.     

The immunocytochemical distribution of seven peptides in the spinal cord and dorsal root ganglia of horse and pig

Summary The distribution of calcitonin gene-related peptide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal polypeptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig.In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concentrated in laminae I–III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of immunoreactive nerve fibres was noted in the lumbosacral segments of the spinal cord, which was particularly exaggerated in the case of VIP immunoreactivity. In the horse, some CGRP-, somatostatin- or tachykinin-immunoreactive cell bodies were present in the dorsal horn. In the pig, cells immunoreactive for somatostatin, enkephalin or NPY were noted in a similar location.In the ventral horn most motoneurones were CGRP-immunoreactive in both species. However, in pig many other cell types were CGRP-immunoreactive not only in the ventral horn, but also in laminae V–VI of the dorsal horn.With the exception of enkephalin and NPY immunoreactivity, which was not seen in pig dorsal root ganglia, all peptides studied were localised to neuronal cell bodies and/or fibres in the dorsal root ganglia. In both species, immunolabelled cell bodies were observed in ganglia from cervical, thoracic, lumbar and sacral levels, with the exception of VIP-immunoreactive cells that were detected only in the lumbosacral ganglia. Numerous CGRP- and tachykinin-immunoreactive cell bodies were visualised in both species, while the cells immunolabelled with other peptide antisera were much lower in number.In both species, immunostaining of serial sections revealed that a subset of CGRP-immunoreactive cells co-expressed tachykinin, galanin or somatostatin immunoreactivity. In the horse some enkephalin-immunoreactive cells were also CGRP positive and occasionally combinations of three peptides, e.g. CGRP, tachykinin and galanin or CGRP, tachykinin and enkephalin were identified.The results obtained suggest that the overall pattern of distribution of peptide immunoreactivities is in general agreement with that so far described in other mammals, although some species variations have been observed, particularly regarding the presence of immunoreactive cell bodies in the dorsal horn of the spinal cord.     

Neuropeptides in interscapular and perirenal brown adipose tissue in the rat: a plurality of innervation

Summary The occurrence of neuropeptides in rat brown adipose tissue has been investigated. Immunohistochemical studies on interscapular and perirenal brown fat have demonstrated unequivocally the presence of substance-P (SP)-like, neuropeptide-Y (NPY)-like and calcitonin gene related-peptide (CGRP)-like immunoreactive elements (putative nerves) in adventitial distribution on inter- and intralobular supply arteries and in accompanying nerve bundles. At a more peripheral level, some NPY-like immunoreactive elements and a greater number of CGRP-like immunoreactive elements were observed in the parenchymal field. Somatostatin, bombesin, neurotensin, enkephalin, and vasoactive-intestinal-polypeptide immunoreactivities were not detected. No differences in neuropeptide distribution were noted between interscapular and perirenal brown fat.There is a degree of coincident distribution of SP, NPY and CGRP with that of noradrenergic nervous elements as visualized by condensation histochemistry. Since after 6-hydroxydopamine treatment not all the nerve terminals in rat brown adipose tissue are stigmatized (earlier report), the present results have been discussed in the light of a possible pluralism in innervation of brown adipose tissue.     

Co-existence of neuropeptides and differential inhibition of vasodilator responses by neuropeptide Y in guinea pig uterine arteries

The co-localization of substance P (SP) with calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP) with neuropeptide Y (NPY) of the guinea pig uterine artery were investigated with immunocytochemistry. The SP/CGRP fibre population was distinct from the VIP/NPY fibre population. Both types of fibres ran in the medial-adventitial border, and appeared as coarsed and fine varicosed. Uterine arterial dilatation was evoked by acetylcholine (ACh), SP, CGRP, and VIP in precontracted arteries as examined by a sensitive in vitro method. Strong relaxations were seen by ACh, CGRP and VIP. NPY had no relaxant effect per se but was found to be a potent inhibitor of vasodilation induced by ACh and SP, while relaxations induced by VIP and CGRP were unaffected. The functional significance of NPY in the uterine artery may to a large extent be to increase tension not only by potentiation of contraction but additionally by inhibiting vasodilator responses.     

Ontogeny of immunoreactive calcitonin gene-related peptide in thyroid C cells from dogs, rabbits, and guinea pigs

Ontogeny of immunoreactive calcitonin gene-related peptide (CGRP) in thyroid C cells of dogs, rabbits, and guinea pigs from early fetuses to adults was investigated by an immunoperoxidase method, in comparison with the development of immunoreactive calcitonin and somatostatin. The presence of immunoreactive CGRP in mature C cells was different from species to species. Dog and rabbit C cells revealed intense immunoreactivity for CGRP, whereas guinea pig C cells revealed very weak immunoreactivity or none. In dog fetuses, the appearance of immunoreactive CGRP was early. At around 35 days of gestation, when the follicular cells were not yet organized into follicles, immunoreactivities for three peptides--calcitonin, somatostatin, and CGRP--began to appear in C cells. While the highest population of somatostatin-positive cells was attained when the primordial follicles were vigorously formed throughout whole thyroid parenchyma and their frequency progressively declined thereafter, CGRP-positive cells as well as calcitonin-positive cells gradually increased in number and intensity with gestational age. The developmental pattern of immunoreactive CGRP coincided with that of immunoreactive calcitonin in dog C cells. In rabbit fetuses, at 25 days of gestation, when thyroid follicles stored large amounts of colloid and C cells already exhibited intense immunoreactivity for calcitonin, CGRP immunoreactivity as well as somatostatin immunoreactivity began to appear. Subsequently, immunoreactivities for the three peptides gradually increased with age, although calcitonin immunoreactivity was outstandingly intense among them. In guinea pig C cells, intense immunoreactivity for CGRP was not observed in any stages of development. These results indicate that there are developmental profiles of CGRP characteristic for each animal, and the ratio of CGRP and calcitonin produced from calcitonin genes in C cells seems to be fixed for life.     

Neurochemical evidence for the involvement of N-type calcium channels in transmitter secretion from peripheral endings of sensory nerves in guinea pigs.

In the guinea pig ureter, substance P-(SP) and calcitonin gene-related peptide-(CGRP) like immunoreactivity (LI) were depleted by systemic capsaicin pretreatment, indicating that they are entirely stored in peripheral endings of primary afferent neurons. Electrical field stimulation (20 Hz, 60 V, 0.5 ms) evoked the simultaneous release of SP- and CGRP-LI from superfused guinea pig ureters which was abolished by tetrodotoxin (0.3 microM). omega-Conotoxin (0.1 microM), a potent blocker of N-type voltage-sensitive calcium channels, reduced by 50-70% the evoked release of both peptides. These findings provide direct neurochemical evidence indicating that conotoxin-sensitive calcium channels play a role in transmitter secretion evoked by antidromic invasion of peripheral terminals of capsaicin-sensitive primary afferents.     

哮喘豚鼠脊髓内CGRP免疫反应的定量分析

应用免疫组织化学和显微图像分析方法，对１６ 只哮喘豚鼠Ｃ７～Ｔ５ 节段脊髓后角内的ＣＧＲＰ免疫反应产物进行了定量研究。结果表明，哮喘豚鼠Ｃ７～Ｔ５ 节段脊髓后角内ＣＧＲＰ阳性反应纤维的密度大大高于正常对照组和实验对照组（Ｐ＜ ０．０１），平均灰度值明显低于正常对照组和实验对照组（Ｐ＜ ０．０１）。本研究结果提示，Ｃ７ ～Ｔ５ 节段脊髓后角内的ＣＧＲＰ可能参与支气管哮喘发病的病理生理过程。本实验结果为进一步探讨哮喘发病的中枢机制提供了一定的形态学依据。     

Expression of interleukin-6 in human dorsal root ganglion cells

The expression of Interleukin-6 (IL-6) was studied in normal dorsal root ganglia (DRG) of juvenile and foetal humans, using immunohistochemistry and in situ hybridization techniques. There was an expression of IL-6-like immunoreactivity in more than 75% out of neuronal cells in the juvenile ganglia with a peripheral localization, and also an expression in the foetal ganglion cells. There was a co-localization of IL-6 with substance P (SP) and calcitonin gene-related peptide (CGRP) in more than 60% of the DRG cells, respectively. By in situ hybridization 0.9% of the cells in the juvenile ganglia and 1.1% of the cells in the foetal ganglia showed a positive signal for IL-6. In addition, expression of IL-6 was found in juvenile medulla spinalis, preferentially in the white matter.     

In situ hybridization studies on mRNAs for cholecystokinin, calcitonin gene-related peptide and choline acetyltransferase in the lower brain stem, spinal cord and dorsal root ganglia of rat and guinea pig with special reference to motoneurons

In situ hybridization techniques were used to analyse the distribution of cholecystokinin (CCK) mRNA in the lower brain stem, spinal cord and dorsal root ganglia of the rat and guinea pig, in comparison with that of mRNAs for calcitonin gene-related peptide (CGRP) and choline acetyltransferase. In the rat, CCK mRNA was found in numerous motoneurons in the spinal cord as well as in the motor trigeminal, facial and hypoglossal nuclei. Coexistence of CCK mRNA and CGRP mRNA could be established in spinal and brain stem motoneurons. Conversely, in the guinea pig CCK mRNA could only be detected in few motoneurons in the spinal cord. In both species, CCK mRNA was present in the spinal trigeminal nucleus and in the dorsal horn of the spinal cord, in numerous small cells located in the outer laminae (mainly II-IV), and in the rat was also found in large cells in laminae IV and V. Few small cells in laminae VI-VIII and X of the spinal cord and cells in several brain stem nuclei, such as the solitary tract, gracile and cuneate nuclei, also showed CCK mRNA in the rat. In the guinea pig brain stem CCK mRNA was found, among others, in the solitary tract nucleus, pontine reticular formation and pontine periventricular grey. In dorsal root ganglia CCK mRNA was abundant in the guinea pig, but almost absent in the rat, where only single cells were found that expressed low levels of this mRNA.     

Distribution and origin of nerve fibers in the rat temporomandibular joint capsule

 The distribution and origin of nerve fibers containing neuropeptides and NOS projecting to the temporomandibular joint capsule (TMJ) of the rat were studied by retrograde tracing in combination with immunocytochemistry. Numerous nerve fibers were seen in the TMJ as revealed by the neuronal marker protein gene product 9.5. Nerve fibers containing neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), substance P (SP), calcitonin gene-related peptide (CGRP), and nitric oxide synthase (NOS) were seen in the synovial membrane, the joint capsule and entering the articular disc. Injection of the retrograde tracer True Blue (TB) into the TMJ resulted in the appearance of numerous labeled nerve cell bodies in the trigeminal and superior cervical ganglia, and moderate numbers in the nodose, the otic, the sphenopalatine, the stellate and the dorsal root ganglia at levels C2–C5. Most of the TB-labeled cell bodies in the superior cervical and stellate ganglia contained NPY. In the trigeminal ganglion, numerous TB labeled cell bodies contained CGRP and a minor population stored SP, a few cell bodies were seen to store NOS or PACAP. In the sphenopalatine and otic ganglia, TB labeled cell bodies contained NOS or VIP. In the nodose ganglion, labeled cell bodies contained CGRP; other labeled cell bodies harbored NOS. In the cervical dorsal root ganglia, the majority of the labeled cell bodies stored CGRP and smaller populations stored SP and PACAP. Thus, the innervation of the TMJ is complex and many different ganglia are involved. Accepted: 13 October 1997     

Co-expression patterns of cocaine- and amphetamine-regulated transcript (CART) with neuropeptides in dorsal root ganglia of the pig

In the present study the neuronal distribution of CART was evaluated immunohistochemically in porcine dorsal root ganglia (DRGs). In co-localization studies the co-expression patterns of CART with SP, CGRP, galanin, CALB and LENK were investigated by means of triple immunohistochemical stainings. In porcine DRGs, the expression of CART was found in approximately 5% of primary sensory neurons. The vast majority (ca. 95%) of CART-immunoreactive (IR) neurons were small and middle sized, and only 5% were categorized as large. CART-IR neurons additionally exhibiting the presence of SP/CGRP (ca. 12%), SP/CALB (ca. 12%), SP/LENK (ca. 5%) were found. The vast majority of CART-IR/CGRP-IR neurons did not display immunoreaction to SP (ca. 60%). Subclasses of CART-IR/LENK-IR/SP-negative (ca. 5%), as well as CART-IR/CALB-IR/SP-negative neurons (ca. 10%), were also visualized. In addition, CART-IR neurons with no immunoreactivities to any of the neuropeptides studied were also shown. In porcine DRGs none of the CART-IR neurons exhibited the presence of galanin. The results obtained in the study suggest that CART may functionally modulate the activity of the porcine primary sensory neurons. It is concluded that co-expression of CART with CGRP, SP, LENK and CALB in subsets of the pig L1-L6 DRGs neurons provide anatomical evidence for a CART role in pain processing.     

Different patterns of immunolocalization of calcitonin gene-related peptide and substance P in sympathetic ganglia of normotensive and genetically hypertensive rats

Calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity were investigated in the superior cervical ganglion of normotensive and genetically hypertensive Otago Wistar rats by an immunoperoxidase method. CGRP- and SP-positive varicose axons invested separate subpopulations of ganglion cells, neither of which contained neuropeptide Y. The densities of CGRP axons were similar in normotensive and hypertensive rats while the numbers of SP axons were several times higher in the hypertensive strain. Decentralization of the ganglion or chronic capsaicin treatment removed all immunoreactive terminals, indicating that both axon populations are likely to be collaterals from thoracic sensory afferents.     

Presence and coexistence of chromogranin A and multiple neuropeptides in Merkel cells of mammalian oral mucosa

By the use of light microscopic (LM) immunohistochemistry, Merkel cells of the mammalian oral mucosa have been examined for the presence and coexistence of some neuropeptides and of the neuroendocrine marker chromogranin A (CG-A). Peptide and CG-A immunophenotypes of oral Merkel cells were found to vary between species and to depend on the developmental stage, as exemplarily revealed in the pig. Oral Merkel cells of adult cat, mouse and pig but not those of adult guinea pig stained for calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI). Pairs of adjacent sections alternately stained for SP, CGRP, VIP, PHI or for CG-A revealed mutual coexistence of these peptides and of CG-A (if expressed) in individual Merkel cells of hard palate, gingiva and buccal mucosa. CG-A immunoreactivity was restricted to Merkel cells of cat and pig. In adult pig and cat, a much lower number of Merkel cells stained for CG-A and peptide expression was inverse. These results indicate that the chemical coding of Merkel cells in mammalian oral mucosa is much more complex than previously described and depends on the developmental stage.     

Peptide-containing nerves in the human pregnant uterine cervix: an immunohistochemical study exploring the effect of RU 486 (mifepristone).

The presence of several neuropeptides (neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), enkephalin (ENK), somatostatin (SOM) was established in the early pregnant human cervix using indirect immunofluorescence immunohistochemistry. Several peptides (VIP, NPY, CGRP, GAL) were present both in free nerves among smooth muscle cells and around blood vessels. Others (SP, SOM) were only seen as single varicosities among smooth muscle cells. Randomized treatment of patients with RU 486 (mifepristone) prior to surgical sampling revealed no clearcut differences in peptide immunoreactivities. After RU 486 treatment, however, there was a tendency towards a decrease of NPY- and VIP-immunoreactivity, and an increase of CGRP-immunoreactivity.     

Simultaneous release of several tachykinins and calcitonin gene-related peptide from rat spinal cord slices

Superfusion of slices of the dorsal half of rat spinal cord in vitro with 10 microM capsaicin or 60 mM potassium lead to the simultaneous release of substance P (SP)-, neurokinin A (NKA)- and calcitonin gene-related peptide (CGRP)-like immunoreactivities (LI). The ratio between capsaicin-stimulated and basal release was higher for CGRP-LI than for SP-LI, indicating that relatively more CGRP is released from sensory nerves, whereas SP is not only released from afferent neurons. High-performance liquid chromatography of NKA-LI revealed several immunoreactive components. One major peak had the retention time of synthetic NKA. A second peak eluted close to the position of synthetic eledoisin. In conclusion, capsaicin releases several bioactive peptides from sensory neurons which may mediate the acute algetic effect of chemical irritants.