Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the teleost cyprinus carpio

Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases, are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. In this work, serial transverse sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of cholinergic distribution pattern in the Cyprinus carpio central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the CNS of the common carp cyprinus carpio

Cholinergic systems play a role in basic cerebral functions and a number of human neurodegenerative disorders. Mechanisms involved in human brain diseases, including Parkinson's disease (1), are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. In this work, serial transverse and sagittal sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei. In particular, ChAT-immunoreactive (ir) neurons were found in the forebrain (preoptic region, habenula), the midbrain (optic tectum, oculomotor nucleus, rostral tegmental nucleus), the hindbrain and the spinal cord (reticular formation, nucleus isthmi, secundary gustatory nucleus, cranial nerve motor nuclei from IV to X, spinal cord motoneurons). Moreover, ChAT-ir neurons were detected in the synencephalon (nucleus of the medial longitudinal fascicle) and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of ChAT-ir distribution observed in the present study with that reported in other CNS of cyprinids (2,3) has revealed a number of similarities and also some interesting differences. Our results provide additional information on the cholinergic system from a phylogenetic point of view, suggesting that cholinergic systems of the common carp show many primitive features that have been conserved during evolution, together with characteristics that are exclusive. In addition, the present study may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases

Endocrine cells distribution in human proximal small intestine: an immunohistochemical and morphometrical study

Atrophy of the pancreatic remnant after pancreaticoduodenectomy might be consequent to deregulation of pancreatic endocrine stimuli after duodenal removal. Relative technical surgical solution could be the anastomosis of the 1st jejunal loop to the stomach and the 2nd to the pancreatic stump. Data on the distribution of endocrine cells within the proximal intestine might represent the lacking tile of the problem. Our aims were to investigate the distribution pattern of serotonin, cholecystokinin and secretin cells in the duodenum, the 1st and 2nd jejunal loops of humans. Bowel specimens of ten patients submitted to pancreaticoduodenectomy were collected; immunohistochemical reactions and morphometric analyses were performed. A general ab-oral decrease of enteroendocrine cells was found. The rate of serotonin cells showed a significant 30.67±8.13% reduction starting from the 1st jejunal loop versus duodenum. The rate of both cholecystokinin and secretin cells in the duodenum was superimposable to that in the 1st jejunal loop, with a significant 62.88±4.80% loss of cholecystokinin and 39.5±9.31% of secretin cells in the 2nd loop. After removal of duodenum, preservation of the 1st jejunal loop could impact the function of pancreatic remnant maintaining the physiological enteroendocrine stimulus for pancreatic secretion that can compensate, at least in part for the abolished duodenal hormonal release

Localization of α-synuclein in teleost central nervous system: immunohistochemical and Western blot evidence by 3D5 monoclonal antibody in the common carp, Cyprinus carpio

Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the CNS of the common carp Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative Western blot, immunohistochemistry and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of ChAT, TH and 5HT by double immunolabelings. Results show that α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. Present findings indicate that α-syn-like proteins may be expressed in few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be co-expressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates. This article is protected by copyright. All rights reserved

Distribution of Tyrosine hydroxylase immunoreactivity in the CNS of the common carp Cyprinus carpio

Catecholamines, including dopamine, are the principal neurotransmitters mediating a variety of functions in the CNS, such as motor control, cognition, emotion, memory processing, and endocrine modulation. Dysfunctional catecholamine neurotransmission is also implicated in neurologic and neuropsychiatric disorders. Human brain diseases, such as Parkinson’s disease (1), have been recently approached by using fish models, especially cyprinid teleosts, given basic similarities of the fish brain to that of mammals. The distribution of the catecholaminergic system has been studied in the forebrain of several teleosts, but relevant information are not available for the common carp, Cyprinus carpio, which is a model species in several studies. In this study, we have analyzed the distribution of catecholaminergic neurons in the carp brain by immunohistochemistry using a specific antibody to tyrosine hydroxylase (TH) on transverse serial frozen sections of the whole brain. In the carp brain, TH-immunoreactive (ir) neurons were present in several nuclei. In particular, positive neurons were detected in the ventral nucleus of the ventral telencephalic area. In addition, neuronal bodies and varicose fibers were stained for TH in the preoptic region, from the anterior to the posterior nuclei, in the suprachiasmatic nucleus, in the ventrolateral and ventromedial talamic nuclei. Moreover TH-ir neurons were also distributed in the periventricular pretectum and locus coeruleus. TH-ir structures were localized not only in recognizable catecholaminergic nuclei, corresponding to those of mammalian brain, but also in regions that are uniquely organized in teleosts, including the ventral telencephalon, the anterior and posterior preoptic region, the ventromedial thalamus, suggesting that they may be useful in elucidating homologies between fish and mammal brain. The present study partially confirmed TH distribution in other CNS of cyprinids (2), and provided more detailed information to a better understanding of the evolution of catecholaminergic system in vertebrates

Synuclein expression in the african clawed frog Xenopus laevis

The synuclein (syn) family comprises three proteins (α-, β- and γ- syns) encoded by different genes (snca, sncb and sncg). In mammals, α- and β- syn are primarily expressed in the brain where they are localized in pre-synaptic terminals while γ-syn is mainly expressed in the peripheral nervous system. In humans, synucleins are involved in neurodegenerative diseases such as Parkinson’s disease and in tumors. However, the normal cellular functions of the three syns have not yet been fully clarified. Members of the syn family were sequenced in representative species of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved, but information about their expression in vertebrate lineages is still scarce. Our research focused on the evolution of syns with the aim of analyzing their molecular and cellular expression in the CNS of representative vertebrates such as the carp Cyprinus carpio for teleost fish (1,2) and the green lizard Anolis carolinensis for reptiles (3). Current model of our comparative analysis for amphibians is the african clawed frog Xenopus laevis. The only information available on syn expression in this species relate to embryonic stages but data on syn expression in the adult are still lacking. At larval stages, amphibian snca is expressed in the brain, branchial arches and somites, and sncb signals were detected in the entire brain and spinal cord whereas sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion (4). Preliminary data are here reported on syn expression in adult specimen of X. laevis, obtained by RT-qPCR, Western blot and IHC. The results demonstrated that syns are expressed both in neuronal and non-neuronal tissues suggesting differences in the expression pattern between developmental and adult stages

Immunoreactivity and expression of synucleins in the South African clawed frog Xenopus laevis peripheral nervous system

Human synucleins (syns) genes coding for α-, β- and γ- isoforms are highly expressed in mammalian nervous system, in particular α-syn is implicated in several neurodegenerative diseases collectively named synucleinopathies, including Parkinson’s disease, frequently associated with motor impairment. The precise functions of syns remain elusive, but there are evidence indicating their involvement in the regulation of vesicular trafficking, exocytosis and synaptic function. Because of the high degree of conservation of syns among vertebrates, non-mammalian animal models may provide additional information on the evolution and the physiological role of these proteins [1,2]. Preliminary data are here reported on α- and β- syns expression and their morphological localization in different organs of adult specimens of the South African clawed frog Xenopus laevis, obtained by RT-qPCR, Western blot (WB) and immunohistochemistry (IHC). In WB and IHC experiments, two different commercial antibodies against mammalian α-, β- syns were used. Alpha- and β-syn immunoreactivities were differently distributed in the various tissues analyzed. Interestingly α -syn immunoreactivity was detected in both peripheral and autonomous nervous system respectively innervating skeletal muscles, cardiovascular system and gastrointestinal tract. Alpha-syn immunoreactive (IR) nerve fibers were found along skeletal muscle fibers, showing large varicosities typical of neuromuscular junctions. Moreover, both submucosal and myenteric plexuses of the gastrointestinal tract showed IR fibers. These preliminary observations suggest a conserved role for α-syn in synaptic vesicle trafficking in peripheral nerves and suggest that Xenopus laevis may be a promising model for the study of synucleinopathies