Amphibian Esophagus Innervation: Study on Neurotransmitters, Slides of Histology

Download Amphibian Esophagus Innervation: Study on Neurotransmitters and more Slides Histology in PDF only on Docsity! Histol Histopath (1 988) 3: 11 5-1 24 Histology and Histopathology lntrinsic and extrinsic innervation of the amphibians esophagic myenteric plexuses C. Junquera, M.J. Azanza, P. Parra, M.T. Peg, J. Aisa, L.M. Romero and P. Garín Cátedras de Biología e Histología, Departamento de Ciencias Morfológicas, Facultad de Medicina, Universidad de Zaragoza, Spain Summary. The innervation of Rana ridibundaesophagus myenteric plexuses has been studied by the following methods: demonstration of cholinesterase activity; F I F method for catecholamines; immunohistochemistry for VIP , S P and S O M , and conventional electron microscopy. The cholinergic innervation is important in the esophagus wall where, in addition to the well known extrinsic component, there is a rich intrinsic plexus with cells and fibres widely distributed. The esophagus, together with the intest ine, a re the Rana gut portions where the adrenergic component is more broadly expressed. T h e adrenergic innervation seems to be almost entirely of extrinsic origin. We have shown that , fo r the tested peptides, there is an intrinsic innervation represented by VIP , S P and S O M like plexuses. W e d o not discard nonetheless an extrinsic component. T h e ultrastructure reveals the morphological characteristics of the enteric neurons as well as the fine inter-relationships between the nervous elements and the functional components of the esophagic wall. Key words: Amphibians, Esophagic innervation, Myenteric plexuses lntroduction Amphibians are interesting from the evolutionary point of view since is in the Anurans where. for the first time in vertebrates, appears a sacra1 parasympathetic system (Burnstock, 1969: Campbell, 1969). In the autonomic system of lower vertebrates the cholinergic system is dominant and in amphibians, for instance, it provides Offprint requests to: Dra. C. Junquera, Cátedra de Biología, Departamento de Ciencias Morfológicas, Universidad de Zaragoza, 50009 Zaragoza, Spain cholinergic and adrenergic innervation to smooth and cardiac muscles. In higher vertebrate forms, the adrenergic system will either replace the primitive parasympathetic system o r will be added to it. It thus seems that. with the increasing vertebrates complexities, the adrenergic sympathetic innervation becomes more important and individualized. With regard to the fibres content in amphibians. both systems. parasympathetic and sympathetic. present a mixed population of cholinergic and adrenergic fibres. The vagus nerve for instance has been considered as a vagus-sympathetic trunk. In the sympathetic pathways the adrenergic fibres proportion is very scarce in amphibians in comparison to the cholinergic one (Boyd et al.. 1964; Norberg et al . , 1967). The relative proportion of the adrenergic fibres in the sympathetic system increases as we approach to mammals. A t the same time a progre5ive substitution at molecular leve1 occurs: the adrenaline, typical of amphibians (Boyd et al., 1964), will be replaced by dopamine and noradrenaline which are typical of mammals (Pscheidt, 1963). Other fibres- classified as non-cholinergic and non-adrenergic have been postulated to be present. For Burnstock those fibres would be purinergic (Burnstock, 1969). Singh suggested the existence of «polinergic» fibres whose synaptic transmitters would change accordingly to the season, as a mechanism of poikiloterms seasonal adaptation (Singh, 1964). O n the other hand, the esophagus intrinsic innervation is represented by the intramural nervous plexuses -myenteric and submucosal in amphibians- which form the Enteric Nervous System (Gershon, 1981). Although it is generally believed that enteric neurons are not found in the submucous plexus of lower vertebrates Wong established, in 1971, the presence of cells in the submucous plexus of both levels, esophagus and stomach. Through their tinctorial characteristici it was considered that they could be either neurons or glial cells. We observed acetylcholinesterase positive cells in the submucous plexus of the frog esophagus and our ultrastructural Amphibians esophagic innervation results shown their neurona1 nature (Junquera et al., 1987b). As well as for the extrinsic component, non- cholinergic and non-adrenergic fibres were postulated to be present in the intrinsic innervation. We have considered that, at least in part. these fibres could be peptidergic. Taking into account the above considerations, the aim of the present work was to study the Rana ridibunda cholinergic, adrenergic and peptidergic (VIP, SP and SOM) esophagus myenteric plexus components. We tried, in this way, to establish the distribution pattern of the cholinergic and adrenergic fibres in comparison with the peptidergic ones and to settle the neurochemical nature of the esophagus extrinsic and intrinsic innervation. At the same time, we shall compare our results with those from other vertebrates in order to establish the phylogenetic differences of the autonomic nervous system at the esophagus level. Materials and methods We have used 20 adult frogs in this study. The general histology of the esophagus wall was examined after staining with haematoxylin and eosin. Acetylcholinesterase method (AChE) Acetylcholine iodide (Sigma) was used for demonstrating the acetylcholinesterase (AchE) activity according to the El Badawi and Schenk (1967) method. The esophagus samples were frozen in methylbutane with liquid nitrogen and 30 pm sections were cut, air dried at room temperature and fixed for 15 min. at 4°C in a solution of 10% formaldehyde (commercial formaline) in PBS (pH 7.0). After washing in distilled water they were incubated for periods of 10-18hr at 37°C. The cholinesterase activity sites were recognized as dark brown precipitates. Controls were made by: (i) incubating in a substrate free medium. and (ii) incubating in a medium with tetraisopropylpyrophosphoramide (ISOONIPA, Sigma). Catecholamines method (FIF) The adrenergic fibres were visualized by the glyoxylic- formaldehyde method (Furness and Costa, 1975). The tissue was fixed by immersion in 2% glyoxylic acid in PBS (pH 7.4) for3hr. It was then frozen in methylbutane with liquid nitrogen, freeze dried overnight or for periods up to 24hr, exposed to paraformaldehyde vapours (3hr at 80°C) and vacuum embedded in paraffin wax. Sections of 15-20 pm were examined under a Leitz-orthoplan fluorescence microscope. Adrenaline produces an apple green fluorescence, while histamine and serotonin produce a yellow fluorescence. lmmunohistochemical methods (IF) We have used the method of indirect immunofluorescence applied on tissue sections and on whole mount preparations. a The method of Coons et al. (1955) has been applied on tissue sections. Esophagus pieces were fixed by immersion in 0.4% p-benzoquinone in PBS (pH 7.1-7.4.) for 3hr at 4" C. They were washed in a 7% sucrose solution in PBS and cryostat blocks were made in methylbutane irnmersed in liquid nitrogen. Sections of 20 pm were incubated in the primary antiserum for 24hr at 4" C in a humid atrnosphere. After washing three times 5min. in PBS the positive reaction sites are recognized by incubating lhr . at room temperature with fluorescein isothiocyanate-conjugated antibodies (FITC, dilution 1:20; Miles Lab. Ltd.). For control, non immune rabbit serum was used as first layer and the FITC globulin was used alone. We have tested the antisera: VIP (dilution 1:200 in PBS; pH 7.2-7.4: INC. Stillwater, MN); substance P (SP, 1:800) and somatostatine (SOM, 1 : 100). We have introduced a modification to the method of Costa et al., 1980) to test the antisera on whole-mount tissue and to examine the three-dimensional distribution of the peptidergic fibres. Pieces of tissue were stretched and pinned on fine cork sheets. The fixation was made by immersion in a solution of 15% saturated picric acid with 2% formaldehyde in 0.1M PBS (pH 7.3) for 18hr at 4OC. After washing in 80% ethanol for 30min. The pieces were dehydrated through a graded series of ethanol and cleared in xylene, 30min. in each solution. The pieces were then rehydrated back to PBS. At this stage was made the delamination. We removed the mucous, submucous and inner muscular layers discovering the contact surface between the two muscle coats: the circular and the longitudinal one. We applied the antisera to both surfaces exposed since the plexuses not always remain attached to the longitudinal musculature. They were incubated for 16hr. at room temperature in a humid chamber. After three washes in PBS the pieces were incubated for lhr . with the conjugated FITC. Then washed in PBS for 15min. and mounted in pure glycerol. The antisera dilutions used were those described above. The slides were kept overnight under pressure. Electron microscopy method The standard method for electron microscopy was applied: fixation in 2.5% glutaraldehyde in Buffer Milloning (pH 7.3). post-fixation in 2% Oso,, stained with 70% uranyl acetate, dehydrated and embedded in araldyte. The ultrathin sections were contrast stained following the conventional methods. Results The haematoxylin-eosin method reveals that the histological organization of the Rana ridibunda esophagus shows some peculiar characteristics. The wall