Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris.

Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

Different sub-cellular localization of alpha-synuclein in the C57BL\6J mouse's central nervous system by two novel monoclonal antibodies.

Alpha-synuclein is a 140 amino acids' protein, widely expressed in the nervous system of different vertebrates and closely related with several neurodegenerative disorders. Although its pathological involvement is reported from long time, its physiological function and its role in neurodegeneration is not yet clear. Disposing of two new monoclonal antibodies, able to detect alpha-synuclein in different compartments of the neurons, the aim of this study is to create an anatomical map of the protein's distribution in the central nervous system of C57 BL\6J mouse, the mouse strain most sensitive to 1-methyl 4-phenyl 1,2,3,6-tetrahydro pyridine neurotoxicity and widely used to apply toxic models of Parkinson disease. The two monoclonal antibodies confirm their ability in visualizing the protein in distinct compartments of the neurons, since 2E3 detects alpha-synuclein in the nerve cells' fibers, whereas 3D5 preferentially in the neuronal nuclei. Both antibodies, instead, are able to show alpha-synuclein at the synaptic terminals. The protein is ubiquitary distributed in the brain, as well as in the spinal cord, but its sub-cellular localization differs markedly in the various regions of the central nervous system. Among alpha-synuclein immunoreactive territories, we describe a particular organization in habenular nuclei, dorsal hippocampus, olfactory bulbs, brain stem nuclei and cerebellar cortex. This preliminary immunohistochemical study, provides the first anatomical map of the alpha-synuclein distribution in the C57 BL\6J mouse CNS and suggests that alpha-synuclein is differentially localized, at sub-cellular level, in different types of neurons and that, therefore, it can plays a specific role for each neuronal subtype. Our study in healthy C57 BL/6J mice represents a starting point to analyze the variations in the overall distribution of alpha-synuclein in mouse models of Parkinson disease.

Immunolocalization of alpha-synuclein in the rat spinal cord by two novel monoclonal antibodies.

This study provides the first immunohistochemical evidence of the presence and distribution patterns in the rat spinal cord of alpha-synuclein (alpha-Syn), a soluble acidic protein, widely expressed in the CNS and closely associated to the pathogenesis of neurodegenerative conditions such as Parkinson's and Alzheimer's diseases. We used two novel homemade monoclonal antibodies (2E3 and 3D5) recognizing two different epitopes of alpha-Syn. Both antibodies localized alpha-Syn within the nerve terminals, whereas 3D5 alone also localized it within the neuronal nuclei. alpha-Syn-immunoreactive nervous elements were widely recognized throughout rat spinal cord and in almost all the gray matter laminae. However, they appeared particularly concentrated within laminae I, II, VII and X and more scattered in the others. Double immunofluorescent labeling showed that alpha-Syn colocalized with synaptophysin in the presynaptic nerve terminals, with neuropeptide Y (NPY) in lamina I, II, IX and X, and had close relationships with tyrosine hydroxylase (TH) immunoreactive neurons in laminae VII and X. Interestingly, the alpha-Syn-immunoreactive nerve elements, in lamina X, contained little of calbindin-28KD and calretinin-31KD. Our findings could help in understanding the genesis of some early clinical symptoms of Parkinson's disease (PD), such as pain and dysautonomic disorders, and indicate the spinal cord as their probable starting point, according to the ascending theory of PD, proposed by Braak.

Endocrine cells in atresic chick embryo intestine: histochemical and immunohistochemical study.

Intestinal motility disorders are an important problem in the postoperative management of patients with intestinal atresia. Intestinal motility could be initiated by luminal factors that activate intrinsic and extrinsic primary afferent nerves involved in the peristaltic reflex. Endocrine cells act as a key point, because they transfer information regarding the intestinal contents and intraluminal pressure to nerve fibers lying in close proximity to the basolateral surface of the epithelium. In chick embryo, experimental intestinal atresia is associated with disorders in the development of the enteric nervous system, related to the severity of intestinal dilation. Our aim was to investigate the distribution pattern of endocrine cells in the developing endocrine system of chick embryo small intestine with experimentally-induced atresia on day 12 and on day 16. Changes in enteroendocrine population were examined in gut specimens (excised proximal and distal to the atresia) from experimental embryos 19 days old and in control sham-operated chick embryos at the same age. Sections from proximal and distal bowel and control bowel were stained with Grimelius silver stain, a valuable histochemical method for detecting the argyrophil and argentophilic cells, and with an immunohistochemical procedure for detecting serotonin and neurotensin immunoreactive cells. In chick embryo proximal bowel, intestinal dilation differed in the various embryos. We found significantly higher enteroendocrine cell counts in proximal bowel than in distal and control bowel. The differences depended on the precociousness of surgery and the severity of dilation. Considering the major contribution of enteroendocrine cells to the peristaltic reflex, our data may help to explain the pathogenesis of motility disorders related to intestinal atresia.

Heroin sensitization induces circumventricular organ activation in the rat brain.

Using c-Fos protein immunohistochemistry we previously demonstrated various sites of activation in the rat forebrain according to the animal's drug history. This study originates from a more detailed evaluation ex-post of the same specimens. A discrete number of c-Fos protein immunoreactive nuclei could be observed in some circumventricular organs, including the vascular organ of terminal lamina (OVLT) and subfornical organ (SFO) and in the nucleus of solitary tract near the area postrema, but only in specimens from sensitized rats. We therefore suggest that repeated drug injections activate the normally low drug metabolizing enzyme activity in the circumventricular organs thus implicating these organs in the complex mechanisms underlying behavioral sensitization.

Distribution of amylin-immunoreactive neurons in the monkey hypothalamus and their relationships with the histaminergic system.

Amylin (AMY) is a 37 amino acid peptide of pancreatic origin that has been localized in peripheral and central nervous structures. Both peripheral and central injection of the peptide causes various effects, including anorectic behavior in rats. Prompted by previous reports showing that the anorectic effect of AMY is mediated by histamine release, we immunohistochemically investigated possible relationships between these two systems at the light microscopical level. Monkey (Macaca fuscata japonica) hypothalamus specimens were submitted to immunohistochemical double staining procedures using AMY and histidine decarboxylase (HDC) antisera. AMY-immunoreactive neurons were found widely distributed in several nuclei of the monkey hypothalamus including the supraoptic, paraventricular, perifornical, periventricular, ventromedial, arcuate, and tuberomammillary nuclei. We detected AMY-immunoreactive nerve fibers throughout the hypothalamus, the median eminence and hypothalamus-neurohypophysial tract. Although AMY- and HDC-immunoreactive neuronal cell bodies occupied distinct hypothalamic zones, many HDC-immunoreactive cell bodies and dendrites, particularly those in the periventricular, arcuate, and rostral tuberomammillary regions, were surrounded by numerous AMY-immunoreactive nerve fiber varicosities. These findings demonstrate for the first time the presence of a discrete number of AMY-immunoreactive neurons in the monkey hypothalamus and add morphological support to the experimental data demonstrating that AMY probably exerts its influence on food intake via the histaminergic system.

Immunohistochemical localization of amylin in rat brainstem.

Immunohistochemical studies were conducted on rat brainstem using a specific polyclonal antiserum against the COOH-terminal (25-37) of human amylin. Amylin-immunoreactive cell bodies were observed in the vestibular, cochlear, trapezoid, and inner cerebellar nuclei and in the mesencephalic nucleus of trigeminal nerve. Positive cell bodies were also found in lateral, gigantocellular and magnocellular reticular nuclei. Numerous amylin-immunoreactive nerve fibers were shown in the trigeminal spinal tract, in the solitary area and in the area postrema. Amylin-immunoreactive cell bodies were often surrounded by a network of tyrosine hydroxylase-immunoreactive nerve fibers. These results provide morphologic evidence that amylin may play a role in some discrete sensory functions.

Expression of guanylin in "pars tuberalis-specific cells" and gonadotrophs of rat adenohypophysis.

The intestinal peptide guanylin regulates the electrolyte/water transport in the gastrointestinal epithelium by paracrine/luminocrine mechanisms. Because guanylin also circulates in the blood, we investigated the rat hypothalamo-pituitary region for expression and cellular localization of this peptide. Reverse transcriptase-PCR analyses with guanylin-specific primers revealed expression of the peptide in the pars tuberalis and pars distalis of the pituitary. Western blotting analyses in hypophyseal tissue extracts identified the expected 12.5-kDa immunoreactive peptide by using two different region-specific guanylin antisera. Light and electron microscopic immunocytochemistry with the same antisera localized guanylin in "pars tuberalis-specific cells" in the juxtaneural pars tuberalis adjacent to nerve endings and blood vessels of the hypothalamo-pituitary portal system and in gonadotrophic cells within the distal pars tuberalis and ventrolateral part of the pars distalis. The presence and cell-specific localization of guanylin within the hypothalamo-hypophyseal system indicate that this peptide may be specifically involved in paracrine and endocrine regulatory mechanisms.

Neuropeptide Y release by pumiliotoxin-B in the electrically-stimulated mouse vas deferens: an immunohistochemical study.

Morphologic and immunohistochemical studies were conducted to ascertain whether pumiliotoxin-B (PTX-B), an indolizine alkaloid from the skin of the Neotropical dendrobatid frog, Dendrobates pumilio, affects the anatomic and immunohistochemical features of the electrically stimulated mouse vas deferens preparations. PTX-B, at a concentration of 1 microM, consistently decreased the density pattern of neuropeptide Y (NPY)-immunoreactive nerve fibers contained within the circular muscular layer. The alkaloid also induced striking morphologic changes. It enlarged the lumen of the vasa and relaxed the muscular wall. Pretreatment with prazosin or haloperidol affected neither the release of NPY nor the morphologic changes; pretreatment with tetrodotoxin and guanethidine abolished NPY release and prevented the PTX-B-induced morphologic changes. PTX-B had no appreciable effect on the density and distribution pattern of nerve fibers immunostained for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, enkephalin, pancreatic polypeptide, 5-hydroxy-tryptamine and tyrosine hydroxylase.

Amylin-immunoreactivity is co-stored in a serotonin cell subpopulation of the vertebrate stomach and duodenum.

Amylin (or islet amyloid polypeptide) is a 37 amino acid peptide originally isolated from amyloid deposits in the pancreas of non-insulin dependent diabetic patients. It has already been immunohistochemically localised within the B and D cells of pancreatic islets and in endocrine cells of the rat and human stomach and duodenum. In this phylogenetic study, a polyclonal antiserum raised against the carboxy-terminal tridecapeptide amide of human amylin was used to demonstrate and examine the distribution of amylin-immunoreactivity in the stomach and duodenum of various vertebrate species. Except for fish, gastrointestinal tracts of all the species studied contained amylin-immunoreactive endocrine cells. They were located chiefly in the lower half portion of the distal gastric body and pyloric glands, and in the lining epithelium of the duodenal villi and crypts. Many cells were elongated, triangular or oval, and had a cytoplasmic process that extended from the cell base along the basement membrane. Others had a bipolar feature that gave them a so-called "open" appearance. Double and triple staining procedures on the same tissue section showed that almost all the amylin-immunoreactive cells present in the gastroduodenal region also co-stored serotonin and chromogranin A, and displayed argyrophilia in Grimelius impregnation. On the other hand, almost all the serotonin-immunoreactive cells of this region co-stored amylin, whereas those in more distal gut regions did not. This finding suggests that those amylin-containing cells correspond to a subtype of gastroduodenal serotonin cells.

Phylogenetic study on distribution and chromogranin/secretogranin content of histamine immunoreactive elements in the gut.

The distribution of histamine-immunoreactive (HA-IR) elements and possible coexistence of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII) were immunohistochemically studied in gut specimens of various vertebrate species. In fish, HA-IR cells were distributed mainly within the gastric and duodenal mucosa, and nerve fibres in the gastric myenteric plexus. Only the gastric HA-IR cells co-stored SgII. In frog specimens, HA-IR endocrine cells and nerve fibres were found in the distal stomach wall, but SgII coexisted only in the nerve fibres. In lizard, HA-IR endocrine cells were widely distributed from the oesophagus to the small intestine, but only those in the stomach co-stored CgA, CgB and SgII. In chick, HA-IR cells were found in the proventriculus and almost all co-stored all three proteins. In rat, HA-IR cells were accumulated in the oxyntic mucosa and all of them constantly immunostained for CgA only.

Phylogenetic aspects of the occurrence and distribution of secretogranin II immunoreactivity in lower vertebrate gut.

A novel monoclonal antibody raised against bovine secretogranin II (Sg II) was used in immunohistochemical studies on amphibian (Rana esculenta), reptilian (Podarcis sicula) and avian (Gallus gallus) gut. Sg II immunoreactivity was detected in epithelial and nervous elements. Cells immunoreactive for Sg II were examined by double immunostainings to determine whether they might also co-store certain previously known bioactive amine/peptide substances. Almost all the endocrine cells immunoreactive for bombesin, substance P, neurotensin, gastrin/cholecystokinin, neuropeptide tyrosine (NPY) and calcitonin gene-related peptide as well as some of those immunostained for serotonin, histamine, and polypeptide tyrosine tyrosine (PYY) also contained Sg II. Sg II-immunoreactive cells varied in number and distribution according to regions of the gut and animal species. The number of Sg II immunoreactive granules notably varied not only according to cell type, but also within the same cell population. Many histamine-, calcitonin gene-related peptide (CGRP)-, substance P-, PYY-, and neurotensin-immunoreactive neurons also contained Sg II. These were mostly situated in the myenteric plexus; their distribution pattern varied among the three species. These findings show that, despite being well conserved during phylogeny, Sg II has a heterogeneous distribution.

Immunohistochemical localization of chromogranin A and B in the endocrine cells of the alimentary tract of the green frog, Rana esculenta.

Novel monoclonal antibodies to human chromogranin A (CgA) and chromogranin B (CgB) were used to investigate the presence of immunoreactive (-IR) elements in the alimentary tract of the green frog Rana esculenta. Numerous CgA-IR and a few CgB-IR endocrine cells were found within the gut mucosa, from the oesophagus to the cloaca, with some local differences in density. Co-localization studies demonstrated that they were co-stored in almost all the serotonin-IR, the amylin-IR or islet amyloid polypeptide-IR cells and in the peptide tyrosine tyrosine-IR cells located proximal to the pylorus, but not in those located in more caudal tracts. No other co-localization was demonstrated; substances investigated included somatostatin, substance P, gastrin/cholecystokinin, glucagon, glycentin, bombesin, secretin and neurotensin. CgA-IR and CgB-IR cells nearly always displayed argyrophilia with the Grimelius silver method.

Sauvagine/urotensin I-like immunoreactivity in the caudal neurosecretory system of a seawater fish Diplodus sargus L. in normal and hyposmotic milieu.

We report the presence of sauvagine/urotensin I-like immunoreactive (SV/UI-LI) elements in the caudal neurosecretory system of a teleost (Diplodus sargus L.) collected from aquaria tanks of the Aquaculture Center (Talassographic Institut of CNR) of Messina or maintained in an hyposmotic milieu for different periods. In normal specimens, SV/UI-LI material was recognizable in discrete or little amounts both in Dahlgren cell cytoplasm and in their axons that reach the urophysis. On the contrary, the specimens transferred in an hyposmotic milieu showed a fast and dramatic increase of immunoreactivity mainly in neurohemal endings of the urophysis. This suggests a physiological role of caudal neurosecretory products on osmoregulatory mechanisms.

Brain-gut-skin peptides: an update overview.

The authors present an overview of the main amphibian peptide families mainly derived from the skin and mostly discovered by Erspamer and his associates. The studies of the peptides do not only promote the understanding of their chemical, metabolical and physiological features of those molecules in amphibians, but also contribute to progress in our knowledge of the corresponding mammalian counterparts. Particular reference is made on sauvagine, tachykinin, bombesin and dermorphin families, offering new data mostly from personal contributions to this field.

Sauvagine/urotensin I-like immunoreactivity in the brain of the dogfish, Scyliorhinus canicula.

The localization of a sauvagine (SV)/urotensin I (UI)-like material in the brain of the dogfish, Scyliorhinus canicula, was studied by immunohistochemical techniques, employing an antiserum raised in rabbit against synthetic SV which widely cross-reacts with UI. Positive cell bodies and nerve fibers were identified in the dorsocaudal hypothalamic region of the tuberculum posterius, in the nucleus sacci vasculosi and nucleus tuberculi posterioris. A dense network of immunoreactive axons was shown in the whole tuberculum posterius. These findings support the view that SV/UI-like peptides may be involved in neuromodulatory functions throughout the brain of cartilaginous fish.

Comparative chemical anatomy of the brain: concepts and methods.

The study of neuropeptides represents an appropriate playground for comparative and evolutionary research. Comparative analysis can give insight into the conservative pattern of intercellular transmission molecules, possibly bound both to some evolutionary antiquity and to cellular constraints. In the same time it can teach us how modulation has occurred at molecular, cellular, multicellular levels in order to give the species-specific functional organization. Using some examples from vertebrate central neurons system (CNS) immunocytochemical analyses, the results so far obtained suggest the rise of a new comparative chemical neuroanatomy. The rationale of "what" and "why" we are comparing is, however, needed in order to understand constancy, heterogeneity or else trends toward complexity in the distribution of neuropeptides.

Ontogenesis of 5-hydroxytryptamine-like immunoreactive cells and their relationship with bombesin in chicken proventriculus.

By using a double-stain immunohistochemical procedure, the Aa. studied the onset, the behaviour and the possible interrelationship between the 5-HT-like and bombesin-like immunoreactive cells during the ontogenesis of chicken proventriculus and in adult animals. The 5-HT-like immunoreactive cells become evident from the 8th day of incubation and they reach their peak around the 16th day, then markedly decrease at hatching. In adult animal these cells are always present, but are not so numerous. Between the 14th and the 18th day many double-stained cells scattered among other only 5-HT or bombesin-like immunoreactive cells are present. They can also be observed in adults, but only occasionally.

Tryptophyllin-like immunoreactivity in rat adenohypophysis.

A new amphibian peptide family has been isolated from the skin of a South American frog Phyllomedusa rhodei and named Tryptophyllins (TPH) because of their content in tryptophyl residue. Using an antiserum against one of these peptides, namely the pentapeptide Met-5-TPH-5-amide (PHE-PRO-PRO-TRP-MET-NH2), we observed the presence of a set of immunoreactive cells in rat adenohypophysis. These cells were far more numerous in pregnant than in normal male and non-pregnant female rats. Dual immunostainings demonstrated that, with some exceptions, almost all the TPH-like immunoreactive cells were gonadotrophs. At electron microscope both types of gonadotroph cells displayed immunoreactivity and the gold particles strongly labelled both types of granules. The Aa. advance the hypothesis that, besides the hormones themselves, the secretory granules might contain some TPH-like sequence.

Active peptides from amphibian skin are also amphibian neuropeptides.

Immunohistochemical data on several newt brain areas showed a heterogeneous system of neuronal elements, immunopositive for antisera specifically directed to different antigenic determinants of tachykinin molecules. Acid extracts of newt brain areas were immunopositive for substance P with an antibody specific for the COOH-terminal (tachykinin determinant) and exhibits an elution profile by gel filtration characteristic to substance P. Bombesin-like immunoreactivity was demonstrable both by immunohistochemistry (fibers and cell bodies in forebrain and hypothalamic areas) and by radioimmunoassay, using an antiserum specific for the mid-region of the molecule. This immunoreactivity was separated into two peaks by gel filtration chromatography. Sauvagine-like material was detected by immunohistochemistry in newt hypothalamus as a loose preoptic cell system and as a thin fiber layer in the outer zone of the median eminence. Double staining procedures demonstrate that sauvagine and CRF are spatially closely related but separable in both the preoptic area and in the median eminence. This data on the presence of several amphibian skin peptides in the CNS of the newt suggests their possible role as neuropeptide.