The distribution of putative neurotransmitters in the nervous system of the dipteran Chironomus tentans insect larva: An immunohistochemical study using antisera to 5-hydroxytryptamine,tyrosine hydroxylase,methionine-enkephalin,proctolin and bombesin

Using the indirect immunofluorescence technique, the localization and distribution of transmitters, transmitter-related enzymes and neuropeptides was studied in the larvae of the dipteran species Chironomus tentans. Immunoreactivity could be seen for 5-hydroxytryptamine, tyrosine hydroxylase (the rate-limiting enzyme in the catecholamine synthesis), and the neuropeptides methionine-enkephalin (met-enk), proctolin and bombesin. The immunoreactivity was confined both to cell bodies as well as to nerve fibers within ganglia and along the alimentary canal. Furthermore, tyrosine hydroxylase immunoreactivity could also be seen in epithelial cells locally distributed along a short, middle part of the alimentary tract. These latter cells were regarded as endocrine-like cells. No immunoreactivity could be found with certainty for the enzyme phenylethanolamine-N-methyltransferase (PNMT) nor for the peptides vasoactive intestinal polypeptide (VIP), dynorphin, substance P, somatostatin, thyrotropin releasing hormone (TRH), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), neurotensin, galanin and cholecystokinin (CCK).     

Sensory and autonomic innervation of non-hairy and hairy human skin

Summary Non-hairy and hairy human skin were investigated with the use of the indirect immunohistochemical technique employing antisera to different neuronal and non-neuronal structural proteins and neurotransmitter candidates. Fibers immunoreactive to antisera against neurofilaments, neuron-specific enolase, myelin basic protein, protein S-100, substance P, neurokinin A, neuropeptide Y, tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) were detected in the skin with specific distributional patterns. Neurofilament-, neuron-specific enolase-, myelin basic protein-, protein S-100-, substance P-, neurokinin A-and vasoactive intestinal polypeptide (VIP)-like immunoreactivities were found in or in association with sensory nerves; moreover, neuron-specific enolase-, myelin basic protein-, protein S-100, neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide (VIP)-like immunoreactivities occurred in or in association with autonomic nerves. It was concluded that antiserum against neurofilaments labels sensory nerve fibers exclusively, whereas neuron-specific enolase-, myelin basic protein- and protein S-100-like immunoreactivities are found in or in association with both sensory and autonomic nerves. Substance P- and neurokinin A-like immunoreactivities were observed only in sensory nerve fibers, and neuropeptide Y- and tyrosine hydroxylase-like immunoreactivities occurred only in autonomic nerve fibers, whereas vasoactive intestinal polypeptide (VIP)-like immunoreactivity was seen predominantly in autonomic nerves, but also in some sensory nerve fibers.     

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.     

Co-localization of peptides in the Brockmann bodies of the cod (Gadus morhua) and the rainbow trout (Oncorhynchus mykiss)

Endocrine cells exhibiting immunoreactivity to FMRFamide-like, LPLRFamide-like, neuropeptide Y(NPY)-like and peptide YY(PYY)-like peptides were found in the periphery of the Brockmann bodies of the cod, Gadus morhua, and rainbow trout, Oncorhynchus mykiss. No immunoreactivity or very weak labelling was found with antisera to pancreatic polypeptide (PP). Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was found in nerve fibres, whereas labelling with VIP antiserum in endocrine cells disappeared after preincubation with nonimmune serum. There were always more immunoreactive cells in the rainbow trout than in the cod. No immunoreactivity could be seen with antisera to gastrin/cholecystokinin (CCK) or enkephalin. Double-labelling studies were performed to study the colocalization of the peptides in peripheral endocrine cells. Cells immunoreactive to NPY were also labelled with antisera to FMRFamide, LPLRFamide and PYY. The co-localization pattern of NPY varied; in some Brockmann bodies, a population of the immunoreactive cells showed co-localization and others contained NPY-like immunoreactivity only, whereas in other Brockmann bodies, all NPY-labelled cells also contained FMRFamide-like, LPLRFamide-like and PYY-like immunoreactivity. Cells immunoreactive to PYY similarly contained FMRFamide-like, LPLRFamide-like and NPY-like immunoreactivity, comparable to the patterns observed with NPY. Glucagon-like immunoreactivity was found at the periphery of the Brockmann bodies. A subpopulation of the glucagon-containing cells contained NPY-like immunoreactivity. PYY-like immunoreactivity was also found co-localized with glucagon-like immunoreactivity, as were FMRFamide-like and LPLRFamide-like immunoreactivity. Therefore, either NPY-like and PYY-like immunoreactivity together with FMRFamide-like and LPLRFamide-like immunoreactivity occur in the same endocrine cells of the Brockmann body of the cod and rainbow trout, or a hybrid NPY/PYY-like peptide recognized by both NPY and PYY antisera is present in the Brockmann body.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin. Relation to fibers containing substance P-, somatostatin- and vasocactive intestinalpolypeptide-like immunoreactivity

Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin gene-related immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Immunohistologic studies of the distribution of proliferative compartments in the appendages of adult human skin

Both, calmodulin (CaM) as well as the antigen Ki67 show a close relationship to cell proliferation. By means of specific antibodies against them, it has become possible to study the spatial distribution of proliferative compartments in tissues. We performed an indirect immunofluorescence study on unfixed frozen sections of human adult skin to gain more informations about the spatial distribution of immunoreactive CaM and Ki67 in skin appendages, i.e. anagen hair follicle, sebaceous and eccrine sweat gland. Two major patterns of immunoreactivity were seen: Type (1) or epidermis-like, which was present in the interfollicular epidermis and the pilosebaceous unit. Type (2) or sweat gland type, which was seen in eccrine sweat glands. Both types disclosed significant differences in the relative number of proliferative cells in S-phase, which might be a consequence of a quiet different tissue architecture. Furthermore, myoepithelial cells of secretory coils were likely to represent mainly SQ-cells. Their immunoreactivity in human skin was quiet different from other parts of eccrine sweat glands suggesting another ontogenetic pathway.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin

Summary Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin generelated immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Vitiligo-related neuropeptides in nerve fibers of the skin

Skin distribution of substance P (SP)-, somatostatin (SOM)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity (LI) in vitiligo patients was studied by an indirect immunofluorescence technique. Immunocytochemical characteristics of the epidermis, dermal-epidermal junction, papillary and reticular dermis and skin appendages were analyzed in lesional and marginal vitiligo areas, as well as in healthy skin. In healthy pigmented skin, SP-, SOM-, CGRP-, and NPY-LI nerve fibers were observed with specific distributional patterns. In uninvolved vitiligo skin, thin SP-containing fibers were evident in dermal papillae, extending into the epidermis, and SP-LI fibers were seen around blood vessels and sweat glands. SOM-LI varicose nerve fibers were associated with Meissner corpuscles in the dermal papillae, while CGRP-LI was demonstrated in the free subepidermal nerve terminals and in sensory nerve fibers around blood vessels, hair follicles and sweat glands. Autonomic NPY-nerve fibers innervated the eccrine sweat glands and blood vessels. The distribution of these neuropeptides in both marginal and lesional areas of vitiliginous skin was the same as in the skin of healthy control subjects, except for an increased immunoreactivity against NPY and, to a lesser extent, against CGRP in the skin depigmentation lesions. The elevated NPY levels in skin affected by vitiligo suggest that this peptide may serve as a neurochemical marker in the pathogenesis of the disease, thus supporting the neuronal theory of vitiligo.     

Presence of proenkephalin in chromaffin cells of the frog adrenal gland

Using a specific antiserum to bovine proenkephalin 1–77 (synenkephalin), the distribution of this peptide in the frog adrenal gland has been studied by means of the indirect immunofluorescence technique. Proenkephalin immunoreactivity was found in all chromaffin cells, which also demonstrated enkephalin- and vasoactive intestinal peptide-like immunoreactivity. No nerve endings containing proenkephalin-, enkephalin-, or vasoactive intestinal peptide-like material could be detected. These data suggest a precursor-product mode of biosynthesis for enkephalins in amphibian chromaffin cells. On a phylogenic point of view, they further indicate a high stability of the structure of proenkephalin during the evolution process.     

The distribution of substance P-, CGRP-, galanin-and ANP-like immunoreactive nerves in human sweat glands

Summary The distribution in immunoreactivities towards atrial natriuretic peptide, calcitonin gene-related peptide, galanin and substance P were demonstrated in human skin at the light and electron microscopic levels. Nerves immunoreactive to the first three of these peptides were found around eccrine sweat glands, whereas only a few positively-labelled nerve fibres could be seen around apocrine glands. At the ultrastructural level, immunoreactivity to the neuropeptides was localized in the large dense-cored vesicles of the nerve terminals. No immunoreactivity to substance P could be detected around sweat glands. In addition to these findings, the four types of immunoreactivity were seen in the thick preterminal nerve bundles.     

Gap junctions in several tissues share antigenic determinants with liver gap junctions.

Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labelling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded.     

LOCALIZATION AND DISTRIBUTION OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE IN THE RAT EPIDIDYMIS

Previous investigation has provided evidence for the control of electrogenic chloride secretion by pituitary adenylate cyclase-activating polypeptide (PACAP) across the rat epididymal epithelium using electrophysiological measurement of transepithelial transport in cultured epididymal system. Hence, it suggests that epididymal and sperm functions are subject to control by a local PACAP system in the rat epididymis. In the present study, localization and distribution of PACAP in the rat epididymal duct was studied by an indirect immunofluorescence technique in conjunction with confocal laser scanning microscopy. Immunoreactivity for PACAP was found in all regions of the epididymal duct. However, the intensity of immunoreactivity for PACAP was stronger in the caput and corpus regions when compared to that of the cauda epididymidis. Much weaker immunostaining for PACAP, as compared to those found in other regions, was observed in the cauda epididymidal tubules which are in close proximity to the vas deferens. No immunoreactivity for PACAP was found in epididymal spermatozoa. Together with the previous finding, the present results suggest that PACAP may exhibit a regional difference in its expression along the epididymal duct and it may act in a paracrine or autocrine fashion in the regulation of epididymal chloride secretion and hence fluid secretion, thus regulating epididymal and sperm functions along the epididymal duct.     

Simultaneous visualization of neuropeptide and acetylcholinesterase nerve subpopulations in the perivascular plexus.

A simple method combining indirect immunofluorescence and histochemical techniques was developed in order to demonstrate the presence of both neuropeptide immunoreactivity and acetylcholinesterase activity in the same whole-mount preparation. It was found that the two methods can be combined without interfering with one another and may be viewed and photographed simultaneously. The guinea pig basilar artery was chosen as a model tissue. While vasoactive intestinal polypeptide immunoreactivity and acetylcholinesterase activity were found to occur in the same perivascular nerve fibres, tyrosine hydroxylase, neuropeptide tyrosine and calcitonin gene-related peptide immunoreactivity were present in distinct nerve subpopulations. It is possible using this double staining procedure, to analyse the interrelationship of putative cholinergic nerves with other components of the autonomic and sensory nervous system.     

Evidence for PHI-immunoreactive nerve fibres in the human skin: coexistence with VIP?

Using indirect immunofluorescence methodology, PHI-like immunoreactivity was found in a certain subpopulation of nerve fibres and terminals of the human skin. The immunoreactive fibres were mainly seen close to and around blood vessels and sweat glands, and they were of a fine-calibre type with smooth preterminal axons and a sparse plexus of varicosities at their terminal field. Furthermore, they were also observed around hair follicles, though more rarely around sebaceous glands. Finally, single PHI immunoreactive fibres could be seen in the close vicinity of the erector pili muscles. These fibres in all probability represent peripheral branches of the autonomic nervous system. Single (somatic?) immunoreactive fibers were, however, also found in the apical parts of the dermis, close to the epidermal-dermal junctional zone. The occurrence of VIP was also analysed and found to be similar to that of PHI. Thus, the present data point to a probable coexistence of PHI and VIP, a possibility that should be taken into account when discussing functional effects of VIP in human skin.     

Lipocortin-1 immunoreactivity in the human pituitary gland

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.     

Peptide hormone-like immunoreactivity in the gastrointestinal tract and endocrine pancreas of eleven teleost species

Summary The distribution of peptide hormone-like immunostaining in the gastrointestinal tract of 11 teleost species was investigated by immunofluorescence.Cells immunoreactive for somatostatin were found in the glandular epithelium of the stomach of four species and in the epithelium of the pyloric appendage of one species. The mid-gut epithelium contained cells reactive with antibodies to glucagon (three species), gastrin (five species), pancreatic polypeptide (five species), and substance P (two species). Cells immunoreactive for met-enkephalin were found in the epithelium of both the mid-gut and the stomach of six species.In six species in which the endocrine pancreas was investigated, insulin-, glucagon-, and somatostatin-like immunoreactivity was observed. Pancreatic polypeptide was definitely localised by immunostaining in cells of the endocrine pancreas of only one out of three species examined.Vasoactive intestinal polypeptide-, neurotensin-, bombesin-, and enkephalin-like immunoreactivity was identified in the gastrointestinal nerve fibres in various species.In view of the considerable species variation found, caution should be exercised in generalising about the peptides present in the gastrointestinal tract of fish.     

Detection of Australia antigen in liver biopsies by immunofluorescence

A search for Australia antigen (AuAg) was made by immunofluorescence in 105 liver biopsies obtained from the same number of patients. No specific fluorescence was observed in 38 cases of acute viral hepatitis (19 of them seropositive for AuAg) or in 55 seronegative patients with various liver disorders or in 8 seronegative patients with histologically normal livers. However, specific fluorescence was seen in two cases: in the single case of chronic aggressive hepatitis seropositive for AuAg and in one of three cases of chronic persistent hepatitis with AuAg-positive sera. The fluorescence observed was mainly intranuclear when cellular suspensions were used, but cytoplasmic fluorescence was more prominent when observations were made on cryostat sections. The finding of AuAg by immunofluorescence in liver cells in chronic but not in acute forms of hepatitis seropositive for AuAg is consistent with the hypothesis of an important role of cellular immunity directed against infected cells in the pathogenesis of viral hepatitis.     

Expression of polysialylated N-CAM during rat heart development

Developmental patterns of immunoreactivity for the neural cell adhesion molecule (N-CAM) and alpha 2.8-linked polysialic acid (PSA) were identified in embryonic and postnatal rat heart by immunocytochemistry and immunoblotting. Polyclonal antibodies against N-CAM and a monoclonal antibody which recognises only polymers of PSA with a chain length greater than eight units were used. Gold- and alkaline-phosphatase-labelled antibodies were used for detection. The N-CAM polypeptide isoform pattern seen by immunoblotting after endoneuraminidase treatment changed as development progressed. During embryonic development a 160-kDa polypeptide isoform was predominant. Around birth, 130-, 160- and 170-kDa polypeptide isoforms were found. The expression of the 130- and 170-kDa isoforms diminished until finally, in the adult, weak immunoreactivity for bands of 120-, 130- and 160-kDa was seen. In general the extent and intensity of PSA and N-CAM immunostaining in rat heart increased until birth and declined thereafter. Early in development prominent immunostaining for PSA and N-CAM was seen in the epicardium while later in development this area was only weakly stained. Initially myocardial cells, endocardial cells and some cells in the atrioventricular cushions were immunoreactive for both PSA and N-CAM. Later in development N-CAM immunostaining was more prominent than PSA immunoreactivity, reflecting a decrease in N-CAM polysialylation, which was also seen by immunoblotting. During innervation of the heart, nerve fibres were strongly immunostained for PSA and N-CAM, and this was the only immunostaining seen in adult heart.     

Regulatory peptides in the gastrointestinal tract of Alligator mississipiensis

Summary The gastrointestinal tract of the alligator Alligator mississipiensis has been investigated for the presence of immunoreactivity to fourteen regulatory peptides all known to occur in the mammalian gut system.Mucosal endocrine cells reacting specifically with the antisera to neurotensin, C-terminal gastrin, somatostatin, bombesin, secretin, pancreatic glucagon and enteroglucagon were detectable, the distribution of these cells being, in general, similar to the mammalian pattern. Peripheral nerve cell bodies and nerve fibres were detected with the antisera to vasoactive intestinal polypeptide, substance P, bombesin and somatostatin again with a distribution similar to that seen in mammals.No immunoreactivity was observed with the available antisera to glicentin, motilin, gastric inhibitory polypeptide, gastrin 34, cholecystokinin 9–20 and met-enkephalin.     

Distribution of peptidergic nerve fibres in bullfrog lingual papillae demonstrated by a combination of double immunofluorescence labelling and a multiple dye filter

Summary Immunoreactivity of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and galanin is localized in nerve fibres distributed in the fungiform and filiform papillae of the tongue of the bullfrog,Rana catesbeiana. A combination of indirect double immunofluorescence labelling and a multiple dye filter system clearly demonstrated that all substance P fibres in the connective tissue core of the fungiform and filiform papillae, and within the rim of ciliated cells located on the top of the fungiform papillae showed coexistence with calcitonin gene-related peptide. A few fibres in the epithelial discs, which are located in the centre of the top of the fungiform papillae, showed the immunoreactivity of calcitonin gene-related peptide alone. There were no substance P fibres which showed coexistence with vasoactive intestinal polypeptide, galanin, and neuropeptide Y. In high magnification images, substance P and vasoactive intestinal polypeptide, and substance P and galanin fibres were recognized as two interwined fibres within the same thin nerve bundle. No immunoreactivity of leucine- and methionine-enkephalins can be detected. These findings suggest that the chemoreceptor function of the bullfrog gustatory organ may be under the control of complicated peptidergic innervation.