Ebe Salvi

Ontogeny of PACAP immunoreactivity in extrinsic and intrinsic innervation of chicken gut☆,1

An immunohistochemical study was conducted on the ontogeny of pituitary adenylate cyclase-activating polypeptide-27 (PACAP) immunoreactive elements within the extrinsic and intrinsic nerve supply of the chicken embryo gut. The first PACAP-immunoreactivity was detected in the extrinsic nerve supply at E 4 within the pharyngeal region and the primary sympathetic chain. At E 5.5 it appeared in the vagus nerve, the spinal cord, the secondary sympathetic chain, some perivascular plexuses and the Remak ganglion. In the intrinsic nerve supply, the first PACAP-immunoreactive elements were shown at E 4.5-E 5 in the mesenchymal bud of the proventriculus/gizzard. Then they gradually appeared also cranially and caudally both in myenteric and submucous plexuses.

AN IMMUNOHISTOCHEMICAL STUDY OF THE ONTOGENY OF THE NEUROENDOCRINE SYSTEM IN THE CHICKEN OESOPHAGUS

Abstract The ontogenesis and distribution of serotonin-, chromogranin A-, chromogranin B-, galanin-, neurotensin-, bombesin- and neuropeptide Y-immunoreactive elements were studied in the chicken oesophagus during pre- and post-hatching life. Unlike positive nerve elements, that were present in pre- and post-hatching life, positive endocrine cells were observed only during embryonic life in the oesophageal epithelium. The first endocrine cells, immunoreactive for serotonin and chromogranins, appeared on day 12, in the cervical and thoracic portions of the oesophagus. At the same age, but only in its distal portion, a few bombesin- and neurotensin-immunoreactive cells also appeared. The number of the endocrine cells progressively increased, reaching a maximum on day 15. They then decreased, with a cranio-caudal progression, until they disappeared a few days after hatching. Almost all the serotonin-immunoreactive cells but only a subpopulation of bombesin- and neurotensin-immunoreactive cells colocalized chromogranins. About half of this subpopulation also colocalized serotonin. All these cells reacted positively with Grimelius argyrophile stain. The mucosa of the crop never contained positive endocrine cells. Positive nervous elements appeared first in the wall of the terminal oesophagus and only one or two days later in the proximal oesophagus including the crop. Nervous elements immunoreactive for galanin first appeared from days 6 to 7, for neurotensin from days 7 to 8, for neuropeptide Y from 13 to 15 and for bombesin from 15 to 18. At day 15 galanin-immunoreactive ganglionic cells and fibres occupied both the myenteric and submucous plexus and galanin-positive nerve fibres could be seen throughout the oesophageal wall from the adventitia to a thin subepithelial network. Neurotensin- and neuropeptide Y-immunopositive ganglionic cells and fibres, by contrast, invariably occupied the muscular and submucous layers. Scattered bombesin-immunoreactive ganglionic cells were observed only in the myenteric plexus. The number of positive nerve elements progressively increased until some weeks after birth. Density and intensity were always much higher for galanin and neurotensin than for neuropeptide Y and bombesin.

Ontogeny, distribution and amine/peptide colocalization of chromogranin A- and B-immunoreactive cells in the chicken gizzard and antrum

The ontogeny and the distribution of chromogranin A (CgA)- and chromogranin B (CgB)-immunoreactive endocrine cells was studied in the chicken gizzard and gizzard-duodenal junction (also called pylorus or antrum) during embryonic and postnatal life. The same tissue sections were then double-immunostained to identify the CgA- and CgB-immunoreactive cells, with a panel of polyclonal antibodies raised against main gut amine/peptides. In the gizzard, positive cells were observed only in its two diverticula (proximal and distal caeca), where the first CgA- and CgB-immunoreactive cells were found on day 12 of incubation. They always remained moderate in number and co-stored mainly serotonin, gastrin/CCK and neurotensin. A few also co-stored somatostatin, but only during the embryonic period. Others co-stored PYY, but only after hatching. Co-localization with motilin was rare and never occurred with bombesin. In the chicken antrum, the first CgA- and CgB-immunoreactive cells were observed on day 12 of incubation and soon reached very high numbers. Antral positive cells showed almost the same co-localization pattern as the gizzard diverticula. Despite their high chromogranin content, the antral cells had weak argyrophilia, whereas in the gizzard diverticula the two staining patterns corresponded.

Spinal cord and parkinsonism: Neuromorphological evidences in humans and experimental studies

The involvement of the spinal cord in parkinsonism is becoming more and more evident based on human autopsies and on experimental models, obtained using specific neurotoxins or genetic manipulations. Besides Parkinson disease, other degenerative disorders characterized by parkinsonism, involve the spinal cord, and multiple neurotransmitters, apart dopamine, are altered in parkinsonism, also in their spinal projections. In the present review we discuss spinal cord pathology of different genetic or toxic experimental models of parkinsonism, as well as the neuropathological reports from autoptic cases of sporadic Parkinson disease and of other neurodegenerative conditions, overlapping with parkinsonism. Furthermore, anatomical distribution of alpha-synuclein in the spinal cord and coeruleo-spinal projections are reviewed, at the light of their possible involvement in spinal neurons degeneration. All these evidences call for an anatomical stemmed novel approach to understand specific features of parkinsonism, which might be due to such an involvement of the spinal cord. Moreover they suggest a common neurodegenerative process, underlying distinct neurodegenerative disorders, to which spinal neurons could be the more sensible.

Ontogeny of galanin-immunoreactive elements in the intrinsic nervous system of the chicken gut.

Galanin is a brain‐gut peptide that is present in the central and peripheral nervous systems. In the gut, it is contained exclusively in intrinsic and extrinsic nerve supplies, and it is involved overall in the regulation of gut motility. To obtain information about the ontogeny of galanin, we undertook an immunohistochemical study of chicken embryos.

Ontogeny, distribution and amine/peptide content of chromogranin A- and B-immunoreactive endocrine cells in the small and large intestine of the chicken

Chromogranin A-(CgA-) and chromogranin B-(CgB-)-immunoreactive endocrine cells were investigated in the chicken intestine during embryonic and post-hatching life. CgA- and CgB-immunoreactive cells first appeared in the intestinal tract at various embryonic ages from day 10 in the cloaca to day 16 in the distal ileum and colon. To identify the CgA- and CgB-immunoreactive cells, each tissue section was double-immunostained using a panel of polyclonal antibodies raised against gut amine/peptides. Almost all the serotonin-immunoreactive cells co-localised CgA and CgB along the entire intestinal mucosa and at all ages examined. In contrast, substance P-, peptide tyrosine tyrosine-, neurotensin- and secretin-immunoreactive cells displayed heterogeneous co-localisation patterns. For example, either all or only some cells of a given endocrine type co-stored Cg; they did so variously-only in the embryo, only after hatching, or at both stages, and co-localizing cells were sometimes located within the mucosa only in the villi and not in the glands, and sometimes vice versa. All the CgA/CgB-immunoreactive cells also displayed argyrophilia.

Ontogeny of galanin-immunoreactive elements in chicken embryo autonomic nervous system

To elucidate the main ontogenetic steps of galanin immunoreactivity within the extrinsic nerve supply of the alimentary tract, we undertook an immunohistochemical study of chicken embryo specimens. Fluorescence and streptavidin‐biotin‐peroxidase protocols were combined, using a galanin polyclonal antiserum, on transverse serial sections obtained from chicken embryos from embryonic Day 3 (E3) to hatching, and from 9‐day‐old newborn chicks. Galanin‐immunoreactive cells were first detected at E3.5 within the pharyngeal pouch region, the nodose ganglion, the primary sympathetic chain, primitive splanchnic branches and the caudal portion of the Remak ganglion. At E5.5 galanin‐immunoreactive cells and fibers appeared in the secondary (paravertebral) sympathetic chain, splanchnic nerves, peri‐ and preaortic plexuses, adrenal gland anlage and visceral nerves. Galanin‐immunoreactive cells also lay scattered along the vagus nerve, and in the intermediate zone of the thoracolumbar spinal cord. At E18, galanin‐immunoreactive cells and fibers were found along the entire Remak ganglion and around the gastrointestinal blood vessels. In post‐hatching‐9‐day old chicks, the para‐ and prevertebral ganglia, but not the intermediate zone of the spinal cord, contained galanin‐immunoreactive cells. Data indicate the presence of a consistent “galaninergic” nerve system supplying the chick embryonal gut wall. Whether this system has growth or differentiating role remains to be demonstrated. Its presence and distribution pattern in the later stages clearly support its well known role as a visceral neuromodulator of gut function. Anat Rec 262:266–278, 2001.

Identification of α-synuclein in the CNS of lower vertebrates by two novel monoclonal antibodies: first immunohistochemical and western blot evidence

Α-Synuclein (α-Syn) is a 140 aa protein which is highly expressed in the CNS of different vertebrates and linked to Parkinson’s disease (PD) in mammals. We have examined the localization of α-Syn in the CNS of zebrafish (Danio rerio), a widely used model species for PD, the carp (Cyprinus carpio) and the African clawed frog (Xenopus laevis) through immunohistochemistry and western blot (WB) by using two new monoclonal antibodies (2E3 and 3D5) against α-Syn. In mammals, both antibodies labeled nerve fibers and 3D5 also localized α-Syn within the neuronal nuclei. Conversely to mammals, no nuclear staining was detectable in fish and amphibian neuronal bodies by 3D5. Moreover, the anatomical distribution of the protein is markedly different. In fact, in mammals α-Syn is ubiquitary distributed in the brain and the spinal cord, whereas in lower vertebrates only few brain regions display a positive staining. In accordance with literature data, these regions provide the amount of dopamine and catecholamines to the brain. Therefore, our results suggest that in lower vertebrates α-Syn is exclusively linked to catecholaminergic and dopaminergic systems and it may represent a protein linked to synaptic vesicles, whereas in mammals it becomes an invariant and structural component of distinct neuronal subtypes assuming a new subcellular localization. WB analysis performed on the CNS homogenates by 2E3 antibody revealed an intense band at 19 kDa. However, the 3D5 antibody stained only faint bands in the same homogenates. These results confirm the presence of α-Syn in the CNS of lower vertebrates analyzed. Our observation suggest that the role of α-Syn in the neuronal physiology could improve, during the evolution of vertebrates and phylogenetic approach could represent a novel method to understand the physio-pahological role of α-Syn in neurological diseases.