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

Synuclein expression in the lizard Anolis carolinensis

The synuclein (syn) family comprises three proteins: alpha-, beta- and gamma-syns. In humans, they are involved in neurodegenerative diseases such as Parkinson's disease and in tumors. 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 and completely lacking in reptiles. In this study, the expression of genes coding for alpha-, beta- and gamma-syns was analyzed in the green lizard Anolis carolinensis by semiquantitative RT-PCR and Western blot. Results demonstrate good expression levels of the three syns in the lizard nervous system, similarly to human syns. This, together with the high identity between lizard and human syns, suggests that these proteins fulfill evolutionarily conserved functions. However, differences between lizard and humans in the expression of syn variants (two different variants of gamma-syn were detected in A. carolinensis) and differences in some amino acids in key positions for the regulation of protein conformation and affinity for lipid and metal ions also suggest that these proteins may have acquired different functional specializations in the two lineages

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

Feeding behaviour of larval European sea bass (Dicentrarchus labrax L.) in relation to temperature and prey density

The feeding behaviour of larval European sea bass (Dicentrarchus labrax, L.) was analysed in relation to temperature and prey density under controlled laboratory conditions with the aim to assess the ability of larval fish to change the feeding tactic as a response to environmental changes. Larvae were acclimated for 20 days at three different temperatures (19, 22 and 26°C), and their feeding behaviour was then video-recorded in experimental trials, at two prey densities, consisting of swarms of 400/l and 1440/l Artemia nauplii. Results showed that there was a significant effect of the interaction between temperature and prey density on the proportion of swimming activity that was reduced at the high temperature-high prey density combination. This suggested a switching in the larval feeding behaviour from an active to an ambush tactic, when the temperature reached 26°C and the prey density was 1440 /l Artemia nauplii. These results are consistent with the current literature on fish larval behaviour in showing that the foraging tactic can be modulated by the interaction of different abiotic and biotic factors characterising the rearing environment

Age and growth determination by skeletochronology in loggerhead sea turtles (<i>Caretta caretta</i>) from the Mediterranean Sea

La esclerocronología se aplicaba a los huesos de los húmeros para determinar la edad y las tasas de crecimiento de la tortuga boba Caretta caretta del Mediterráneo. Cincuenta y cinco tortugas bobas muertas de 24 a 86.5 cm de longitud de la curvatura del caparazón (CCL) fueron recogidas del Mediterráneo central. Secciones de los húmeros fueron procesados histológicamente para analizar las marcas anuales de crecimiento. Se aplicaron dos aproximaciones para determinar el crecimiento somático utilizando la función de crecimiento de von Bertalanffy. La primera aproximación se basaba en el número total de marcas de crecimiento, correspondiendo a la edad de las tortugas en el momento de la muerte. La segunda aproximación era una estimación de la longitud del caparazón en las marcas de crecimiento más antiguas, con la intención de saber la tasa de crecimiento de cada tortuga. Las tasas de crecimiento individual observadas, oscilaban entre 1.4 y 6.2 cm año–1, mostrando una elevada diversidad individual intra e interanual, posiblemente ligada a la variabilidad ambiental experimentada por las tortugas bobas durante su vida. Ambas aproximaciones dieron resultados similares y sugieren que la tortuga mediterránea tarda 14.5-28.5 años en alcanzar un tamaño de 66.5-84.7 cm de CCL. Este tamaño corresponde al tamaño medio de las tortugas bobas hembras nidificantes, encontradas en la mayoría de lugares nidificantes del Mediterráneo y puede ser considerado el tamaño aproximado de la madurez

Determinación de la edad y el crecimiento por esclerocronología en la tortuga boba marina Caretta caretta del mar Mediterráneo

Skeletochronology was applied to humerus bones to assess the age and growth rates of loggerhead sea turtles (Caretta caretta) in the Mediterranean Sea. Fifty-five dead turtles with curved carapace lengths (CCL) ranging from 24 to 86.5 cm were collected from the central Mediterranean. Sections of humeri were histologically processed to analyze annual growth marks. Two approaches were used to estimate the somatic growth in the form of a von Bertalanffy growth function. The first approach was based on calculating the total number of growth marks, which corresponds to the age of turtles at death. The second approach estimates the carapace length at old growth marks in order to provide the growth rate of each turtle. The observed individual growth rates ranged from 1.4 to 6.2 cm yr–1, and showed both elevated inter- and intra-individual variability possibly related to the environmental variability experienced by turtles during their lifetime. Both approaches gave similar results and suggest that Mediterranean loggerhead turtles take 14.9 to 28.5 years to reach a CCL of 66.5 to 84.7 cm. This size corresponds to the average size of nesting females found in the most important Mediterranean nesting sites and can be considered the approximate size at maturity.La esclerocronología se aplicaba a los huesos de los húmeros para determinar la edad y las tasas de crecimiento de la tortuga boba Caretta caretta del Mediterráneo. Cincuenta y cinco tortugas bobas muertas de 24 a 86.5 cm de longitud de la curvatura del caparazón (CCL) fueron recogidas del Mediterráneo central. Secciones de los húmeros fueron procesados histológicamente para analizar las marcas anuales de crecimiento. Se aplicaron dos aproximaciones para determinar el crecimiento somático utilizando la función de crecimiento de von Bertalanffy. La primera aproximación se basaba en el número total de marcas de crecimiento, correspondiendo a la edad de las tortugas en el momento de la muerte. La segunda aproximación era una estimación de la longitud del caparazón en las marcas de crecimiento más antiguas, con la intención de saber la tasa de crecimiento de cada tortuga. Las tasas de crecimiento individual observadas, oscilaban entre 1.4 y 6.2 cm año–1, mostrando una elevada diversidad individual intra e interanual, posiblemente ligada a la variabilidad ambiental experimentada por las tortugas bobas durante su vida. Ambas aproximaciones dieron resultados similares y sugieren que la tortuga mediterránea tarda 14.5-28.5 años en alcanzar un tamaño de 66.5-84.7 cm de CCL. Este tamaño corresponde al tamaño medio de las tortugas bobas hembras nidificantes, encontradas en la mayoría de lugares nidificantes del Mediterráneo y puede ser considerado el tamaño aproximado de la madurez

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