Monoamines in the Pedal Plexus of the Land Snail Megalobulimus Oblongus (Gastropoda, Pulmonata)
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Brazilian Journal of Medical and Biological Research (2004) 37: 1043-1054 Monoamines in the pedal plexus of Megalobulimus oblongus 1043 ISSN 0100-879X Monoamines in the pedal plexus of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata) M.C. Faccioni-Heuser1, 1Laboratório de Histofisiologia Comparada, Departamento de Ciências Morfológicas D.M. Zancan2 and 2Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde, and M. Achaval1 Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil Abstract Correspondence In molluscs, the number of peripheral neurons far exceeds those Key words M. Achaval found in the central nervous system. Although previous studies • Pedal plexus Laboratório de Histofisiologia on the morphology of the peripheral nervous system exist, details • Catecholamines Comparada, Departamento de of its organization remain unknown. Moreover, the foot of the • Serotonin Ciências Morfológicas, ICBS, UFRGS terrestrial species has been studied less than that of the aquatic • Glyoxylic acid Rua Sarmento Leite, 500 • species. As this knowledge is essential for our experimental model, Immunohistochemistry 90050-170 Porto Alegre, RS • Gastropod Brasil the pulmonate gastropod Megalobulimus oblongus, the aim of the Fax: +55-51-3316-3092 present study was to investigate monoamines in the pedal plexus E-mail: [email protected] of this snail using two procedures: glyoxylic acid histofluorescence to identify monoaminergic structures, and the unlabeled antibody Research supported by FINEP peroxidase anti-peroxidase method using antiserum to detect the (No. 66.91.0509.0) and FAPERGS. serotonergic component of the plexus. Adult land snails weighing 48-80 g, obtained from the counties of Barra do Ribeiro and Charqueadas (RS, Brazil), were utilized. Monoaminergic fibers were Received May 30, 2003 detected throughout the pedal musculature. Blue fluorescence Accepted April 13, 2004 (catecholamines, probably dopamine) was observed in nerve branches, pedal and subepithelial plexuses, and in the pedal muscle cells. Yellow fluorescence (serotonin) was only observed in thick nerves and in muscle cells. However, when immunohistochemical methods were used, serotonergic fibers were detected in the pedal nerve branches, the pedal and subepithelial plexuses, the basal and lateral zones of the ventral integument epithelial cells, in the pedal ganglion neurons and beneath the ventral epithelium. These findings suggest catecholaminergic and serotonergic involvement in locomotion and modulation of both the pedal ganglion interneu- rons and sensory information. Knowledge of monoaminergic dis- tribution in this snail´s foot is important for understanding the pharmacological control of reflexive responses and locomotive behavior. Introduction nervous system (1,2). The foot is one of the most innervated areas in molluscs. The periph- In many invertebrates, especially in mol- eral nervous system of the foot includes a pedal luscs, the number of peripheral neurons can far plexus, which is involved in locomotion, exceed the number of neurons in the central body movement and sensory function (1,2). Braz J Med Biol Res 37(7) 2004 1044 M.C. Faccioni-Heuser et al. A high degree of structural organization modulatory and premotor interneuron cells. has been described in the epithelial and mus- A detailed analysis of the structure and chem- cular areas of the foot sole in Megalobulimus ical nature of the neuronal circuits is a nec- oblongus (3). The nerves from pedal ganglia essary prerequisite of neurophysiological give rise to small nerves that branch out to studies. The usefulness of unique properties form the pedal plexus. This plexus consists of molluscan nervous system for neurobio- of a nerve meshwork with ganglionic clus- logical research is widely recognized. The ters located among the pedal muscle fibers. involvement of the serotonergic and dopa- A similar organization has been described in minergic neuronal networks in aversive, feed- other gastropods, such as Helix aspersa (4) ing, locomotive, and respiratory behavior and Ereminia ehrenbergi (5). has been described in other gastropods. The distribution of neurotransmitters in Given the absence of studies on the dis- the pedal plexus of some aquatic molluscs tribution of monoaminergic systems in the has been described. Neurotransmitters such peripheral nervous system of terrestrial gas- as acetylcholine, dopamine and serotonin (5- tropods and the importance of this knowl- HT), diverse neuropeptides such as sub- edge for our experimental model, M. oblon- stance P and calcitonin gene-related peptide gus, the aim of this study was to investigate have been identified (3,6,7). There is some monoamines in the pedal plexus of this snail disagreement regarding the presence of using the glyoxylic acid fluorescence proce- monoaminergic or peptidergic neuronal so- dure and immunohistochemistry to detect mata in the foot (8). 5-HT. Among invertebrates, the most common catecholamine is dopamine, while adrenaline Material and Methods and noradrenaline are rare (6). In gastro- pods, dopamine is also the principal cate- Adult land snails, M. oblongus - Müller cholamine and there is no evidence that the 1774, weighing 48-80 g were obtained from neurons are capable of synthesizing other the counties of Barra do Ribeiro and catecholamines (6,9). In a pharmacological Charqueadas (RS, Brazil) over a period of review of rhythmic behaviors such as loco- one year. The animals were kept in a screened motion or breathing, the modulatory role of terrarium, subjected to a controlled 12-h 5-HT is noteworthy in the control of locomo- light/dark cycle and temperature (20-25ºC) tion (8,10) and feeding (11-13), while dopa- and fed lettuce and water ad libitum. mine has been reported to play a role in breathing (14). In addition, the modulatory Histofluorescent method role of 5-HT in aversive behavior (10,15) has also been documented (16). The histofluorescence method of De La Most of the available knowledge con- Torre and Surgeon (18), with some modifi- cerning control of locomotion behavior comes cations (19), was used to detect monoam- from studies of aquatic gastropod species ines. Briefly, 23 snails were anesthetized by (ciliary gliding or swimming gastropods). immersion in a menthol-saturated physiologi- However, terrestrial molluscs, which use cal solution (29.5 mM NaCl, 2.4 mM KCl, waves of pedal muscular contraction for 6.0 mM CaCl2) for 30 min. The foot muscu- crawling, have been studied less (17). lature was dissected out and samples of the One of the most difficult problems in anterior, medial and posterior regions of the neuroscience is the functional identification pedal muscle were obtained (20). The tissue of the neurons involved in the networks fragments were frozen and horizontally or underlying certain behavior, specially the transversely sectioned (75 µm) with a cry- Braz J Med Biol Res 37(7) 2004 Monoamines in the pedal plexus of Megalobulimus oblongus 1045 ostat (Leitz Digital 1702) and picked up on cessed for 5-HT immunohistochemistry by glass slides. The sections were incubated in the unlabeled antibody peroxidase-antiper- a solution containing 2% glyoxylic acid, 0.2 oxidase procedure (24). The sections were M sucrose and 0.236 M monobasic potas- pretreated with 10% methanol diluted in 3% sium phosphate diluted in distilled water at hydrogen peroxide for 30 min, carefully pH 7.4 for 3-5 min. The incubation in this washed and blocked with 3% normal goat solution and the subsequent steps were per- antiserum in PBS containing 0.4% Triton- formed according to the method of Salimova X100 for 30 min. The free-floating sections and colleagues (21). The sections were ex- were incubated with a polyclonal antiserum amined with a Nikon fluorescence micro- raised in rabbits against 5-HT (Sigma) diluted scope, Optiphot-2, fitted with a V-2A filter 1:2000 in PBS-Triton X100 with continuous block (BA450 barrier filter and EX 380-425 shaking for 48 h at 4ºC. The sections were excitation filter), giving blue fluorescence to washed in PBS and incubated with anti- catecholamine fluorophores and yellow flu- rabbit IgG (1:50; Sigma) for 2 h at room orescence to indole-reactive products. temperature. The sections were washed in Two control procedures were performed. PBS and incubated with a rabbit peroxidase- In the first, 5 snails were injected into the antiperoxidase antibody (Sigma) diluted 1:500 anterior part of the foot (22) with 30 µg/g for 2 h at room temperature. In order to reserpine phosphate (Ciba) dissolved in a develop the immunohistochemical reaction, snail saline solution. The animals were kept at the sections were treated in a medium con- room temperature and after 3 days were taining 0.06% 3,3'-diaminobenzidine tetra- processed as previously described. In the hydrochloride (Sigma) for 10 min at room second, glyoxylic acid was omitted from the temperature and then in the same solution reaction solution used for sections from the containing 1 µl 3% H2O2 per ml for 10 min. pedal muscle. For control, sections from the different re- gions of the pedal muscle were routinely Immunohistochemistry processed by omitting the primary antibody. This study was performed on 8 animals. Results Two snails were injected intramuscularly with 10 mg/g 0.1% colchicine (Enila Labora- The histochemical glyoxylic acid method tory) (23) 48 h before muscle removal. The labeled monoaminergic fibers of varying di- animals were then anesthetized in a menthol- ameters as well as the pedal plexus, located saturated physiological solution and samples along the pedal muscle of the foot of M. from the anterior, medial and posterior re- oblongus. Both the same strong blue fluores- gions of the pedal muscle (20) were obtained cence of the catecholamines and the intense and fixed in a fresh solution containing 4% or pale yellow fluorescence characteristic of (v/v) paraformaldehyde in 0.1 M sodium 5-HT were observed throughout the pedal phosphate buffer, pH 7.4, at room tempera- muscle (Figure 1a,b). The 5-HT fibers quickly ture for 4 h. The samples were then cryopro- faded when exposed to ultraviolet light.