An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Innervation of the gastrointestinal canal of the toad Bufo marinus by neurons containing 5-hydroxytryptamine-like immunoreactivity

Summary The gut of the toad, Bufo marinus, was examined for evidence of enteric neurons containing 5-hydroxytryptamine-like immunoreactivity. Such neurons were absent from the stomach. They were present in the small intestine, with processes confined to the myenteric plexus. Immunoreactive nerve cell bodies lay on branches of the pelvic nerves supplying the large intestine; fibres were found in the submucosa of the posterior large intestine and in the muscularis externa of the anterior large intestine. It is concluded, on morphological grounds, that the neurons in the small intestine are interneurons, whereas those in the large intestine are postganglionic parasympathetic motoneurons.     

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad,Bufo marinus

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galamin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.     

Membranes during yolk-platelet development in oocytes of the toad Bufo marinus

Summary Oocytes of the toad Bufo marinus have been studied by means of thin section and particularly freeze-fracture electron microscopy to characterize the cytoplasmic membranes around the yolk organelle, and the storage of yolk material in precursors and platelets. This appears to be a previously unknown type of yolk-platelet formation. During yolk-organelle development from the primordial precursor to the bi-partite fully grown yolk platelet, numerous lipoid droplets are attached to the periphery of the platelet, indicating an intense uptake of lipids. As is typical for amphibians, the fully grown yolk platelet has a crystalline internum covered by a dense osmiophilic externum, and the whole organelle is enveloped by a plasma membrane that shows no direct connection or fusion with endocytotic vesicles. The yolk membrane exhibits few intramembraneous particles (IMPs) at the core areas and some more where it borders fields of lipoid droplets. Here the IMPs show a net-like arrangement in the furrows between adjacent droplets.     

The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus)

Summary The cytological features and membrane specialisations of neuroepithelial cells (apical cells) in direct contact with the lumen of the lung were studied with transmission and scanning electron microscopy. The luminal surface of the apical cell is characterised by microvilli, a cilium with an 8+1 microtubular pattern and numerous coated vesicles. The cytoplasmic region immediately beneath the luminal plasma membrane contains numerous smooth-walled vesicles, tubules and microtubules, a few microfilaments and dense granules (15–20 nm in diameter). The luminal pole of the cell is marked off from the basal or vascular pole by a well-defined terminal web associated with junctional complexes. Protrusion of the luminal pole occurs as a transient phenomenon and is accompanied by a pinching in of the cell at the terminal web. It is proposed that the distinctive features of the luminal pole of the apical cell are comparable to those of recognised chemoreceptor cells. It is also proposed that in view of the common features of apical and basal cells the apical cell functions as a receptor/transducer and the basal cells serve as an accessory source of peptides/5-hydroxytryptamine to be released on stimulation of the apical cell. Furthermore, we have drawn attention to the structural heterogeneity of the neuroepithelial bodies in various vertebrate classes.     

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad,Bufo marinus

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.     

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus)

Summary Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibres containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which form distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P_{CO 2} and so leading to modulation of activity in afferent nerve fibres (type containing agranular vesicles). The presence of NEBs solely with an adrenergic (efferent) innervation poses a problem with this interpretation.     

Geographical patterns of genetic variability in introduced Australian populations of the marine toad,Bufo marinus: Sorbitol dehydrogenase,Sdh

The marine toad, Bufo marinus, was introduced to Australia from Hawaii in 1935. From 1935 to 1974, the toad population expanded exponentially to occupy 584,000 km², and now has a continuous distribution from Cape York to Tweed River on the eastern coast of the continent. Genetic analysis of the population indicates a difference in allele frequency at the sorbitol dehydrogenase locus. There are two alleles segregating at the locus (NAD-Sdh^a and NAD-Sdh^b). The NAD-Sdh^aa homozygote is common in the two southern populations, but uncommon in northern populations. The north-south difference has been established in less than 25 generations.     

Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons

Summary The innervation of the major arteries and heart of the toad (Bufo marinus) was examined by use of glyoxylic acid-induced catecholamine fluorescence and peptide immunohistochemistry. All arteries possessed a moderate to dense plexus of adrenergic axons, which also showed neuropeptide Y-like immunoreactivity (NPY-LI). Some adrenergic axons in the intracardiac vagal trunks showed NPY-LI, but the varicose adrenergic axons innervating the cardiac muscle of the atria and ventricle, and the coronary blood vessels did not display NPY-LI. About half of the nerve cell bodies in the anterior sympathetic chain ganglia with dopamine--hydroxylase-LI (DBH-LI) also contained NPY-LI. The nerve cell bodies with DBH-LI alone were generally larger (median diameter 30 m) than those with both DBH-LI and NPY-LI (median diameter 20 m). Some cell bodies showing DBH-LI alone were surrounded by boutons with NPY-LI but not DBH-LI. Axons that displayed simultaneously both substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI) also formed a plexus around all arteries studied, being particularly dense around the mesenteric and pulmonary arteries. These axons are most likely sensory since SP-LI was reduced by capsaicin treatment, and nerve cell bodies with both SP-LI and CGRP-LI were found in dorsal root ganglia and the vagal ganglion. A dense plexus of axons showing somatostatin-LI was located around the pulmonary artery and its main intrapulmonary branches. A few nerves with vasoactive intestinal polypeptide-LI were found around the dorsal aorta and pulmonary artery. No perivascular nerves with enkephalin-LI were observed. Reversed-phase, high-pressure liquid chromatography of acid extracts of the large arteries showed that the major peaks of NPY-LI and SP-LI coeluted with porcine NPY (1–36) and synthetic SP (1–11), respectively. Thus, the location and structure of these peptides in perivascular nerves has been highly conserved during vertebrate evolution.     

The distribution of 5-hydroxytryptamine in the gastrointestinal tract of reptiles,birds and a prototherian mammal

Summary The distribution of 5-hydroxytryptamine in the gut of several species of birds and reptiles, and of a prototherian mammal, the platypus, was studied using a monoclonal antibody. 5-Hydroxytryptamine-like immunoreactivity was found in enterochromaffin cells and, in birds, in thrombocytes. Immunoreactivity was not found in enteric neurons fixed immediately after dissection. A detailed study was made on one avian species, the budgerigar. Following incubation of intestine in physiological solution, immunore-activity was found in nerve fibres in the gut wall that was more marked after incubation with the monoamine oxidase inhibitor pargyline. These fibres took up exogenous 5-hydroxytryptamine. Similar fibres were found in the intestinal nerves and in perivascular plexuses on mesenteric arteries. Both the uptake of 5-hydroxytryptamine and the appearance of neuronal immunoreactivity after incubation were inhibited by the amine uptake inhibitors desmethylimipramine or fluoxetine. Fibres taking up 5-hydroxytryptamine were damaged by pretreatment with 6-hydroxydopamine. It was concluded that the fibres showing immunoreactivity after incubation were adrenergic fibres that had taken up 5-hydroxytryptamine released in vitro from enterochromaffin cells or thrombocytes. These, and more limited observations made on the other species, suggest that birds, reptiles and prototherian mammals lack enteric neurons that use 5-hydroxytryptamine as a transmitter substance.     

Fine structural and cytochemical study of the innervation of smooth muscle in an amphibian (Bufo marinus) lung before and after denervation

Summary The innervation of the toad (Bufo marinus) lung was studied with transmission electron microscopy and fluorescence techniques, both before and after 12 or 20 days close vagosympathetic denervation. Four cytologically distinct types of neuronal processes were recognised, in relation to the visceral muscles of the lung. These were described as cholinergic, adrenergic, nonadrenergic/non-cholinergic (NANC) and sensory on the basis of the characteristics of their vesicular content and cytochemical reactions. An apparent efferent innervation of visceral smooth muscle was achieved by NANC (50%), cholinergic (25%) and adrenergic (25%) fibres. A few sensory fibres were also present. After denervation only NANC fibres persisted, showing that the cell bodies of these fibres were intrapulmonary. The vascular smooth muscle was supplied by cholinergic, adrenergic and sensory fibres. In the walls of the proximal branches of the pulmonary artery were fibres containing large dense-cored vesicles. These profiles, which were associated with the vasa vasorum, were similar to neurosecretory fibres. After denervation all neural profiles associated with the vasculature had degenerated. The observations suggest that vagal vasodepressor effects in the toad lung are mediated indirectly through relaxation of visceral muscle strands which in their contracted state compress vascular channels.The authors would like to thank Dr. J.R. McLean for technical advice on fluorescence microscopy. This work was supported by a grant from the Australian Research Grants Committee     

Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary     

Evidence for the existence of serotonin-, dopamine-, and noradrenaline-containing neurons in the gut of Lampetra fluviatilis

Summary Monoamine-containing neurons in the gut of Lampetra fluviatilis are characterized by histochemical, electron microscopical and biochemical methods. Strongly yellow fluorescent, probably serotonin-containing intrinsic neurons are found along the entire length of the intestine. Their processes aggregate to form large bundles of mainly non-terminal axons, constituting a subepithelial fibre plexus. This subepithelial, ganglion cell comprising plexus is connected to a wide-meshed subserosal plexus which has ganglion cells of different size and few varicose, single axons. Intermingled with both plexus there occur — in the anterior and middle but not in the preanal portion of the lamprey intestine — scattered green fluorescent intrinsic perikarya, emanating faintly green fluorescent, poorly varicosed axons.The formaldehyde-induced neuronal fluorophores conform to serotonin (yellow fluorescent compound), noradrenaline, and dopamine (green fluorescent substance), as revealed in microspectrofluorimetric recordings. The electron microscopical analysis of the yellow fluorescent intrinsic neurons in the terminal hindgut shows nerve cell pericarya and axons equipped with a typical population of occasional small granular and many large granular vesicles (750–1600 Å). The number and opacity of cores of the small and the osmiophilia of the cores of the large granular vesicles are significantly increased following short-term treatment with 5,6-dihydroxytryptamine. Long-term treatment with 5,6- or 5,7-dihydroxytryptamine provokes severe signs of ultrastructure impairment and eventual degeneration in the supposed serotonin-containing axons, besides indications of piling-up of organelles in the non-terminal axons due to arrest of axonal transport.Chromatography of acid extracts from the lamprey intestine, gills and kidney reveals the presence of serotonin (besides another unidentified indoleamine) and dopamine and noradrenaline in the gut, but only dopamine in the brain. The detection of serotonin, noradrenaline and dopamine in the lamprey gut is confirmed by chemical determinations.The occurrence of intrinsic serotonin-, noradrenaline- and dopamine-containing neurons in the gut of Lampetra fluviatilis deviates from the established pattern of innervation of the vertebrate intestine and is considered to be a remnant of an autonomic innervation principle common in invertebrates.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by grants from the Swedish Medical Research Council (No. 14X-712 and 14X-56.The authors are indebted to Lilan Bengtsson, Gertrude Stridsberg, Eva Svensson and Rolf Frank for skilful technical assistance.     

Temporal spacing of breeding activity in the natterjack toad,Bufo calamita

Summary The reproductive behaviour of the toad Bufo calamita was studied in a large population in the northern Rhineland, Germany. Toads bred at four spatially separated but neighbouring areas including 34 breeding ponds. Three methods were applied to monitor quantitatively the intensity of breeding and migratory activity: capture-mark-recapture, a mechanical tracking device, and radio-telemetry. In 1987 a twofold temporal spacing of breeding activity was observed: (1) division of the reproductive period into early, main, and late breeding periods; (2) subdivision of each breeding period into several short calling periods. Since weather conditions were favourable during most of the reproductive period, environmental factors contributed little (23.4%–41.0%) to the temporal variation in breeding intensity as shown by multivariate statistics. The period of vocalization (April to August) included the activity of three distinct male groups. The first group called from April to mid-May (2 calling periods), the second from May to early July (5 calling periods) and the third from mid-July to August (2 calling periods). Within a calling period most males stayed near their calling sites and migratory activity was low. Extensive movements were detected when a new group of males immigrated from the feeding regions to the breeding areas and males that had been calling previously emigrated, at least partly. However, exchange of males among the neighbouring breeding choruses were clearly audible. It is suggested that the energetic costs of vocalization impeded continuous calling activity in individuals and accounted for the periodical variations in chorus size. The short-time variations of breeding activity therefore probably reflect the physiological limits of toads in a situation where the weather permits continuous reproductive activity.     

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus)

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.     

Exocytosis of cortical granules from activated oocytes of the toad,Bufo arenarum

Summary Observation of the cortical region of oocytes of Bufo arenarum by transmission electron microscopy reveals modifications on their surface and in the contents of the cortical granules (CG) during activation. In non-activated oocytes only amorphous cortical granules (ACG) can be observed. Activated oocytes display ACG, intermediate cortical granules containing both amorphous and membranous material (ICG), and a third type containing only membranous material (MCG). During exocytosis, CG release their contents into the perivitelline space, where the amorphous and membranous materials are found. The three types of CG found during oocyte activation suggest transformation of ACG to MCG and indicate that the different components of the cortical granules, when released into the perivitelline space, might play different roles in prevention of polyspermy.Members of the Scientific Research Career of CONICET, R. Argentina.     

Effects of the ecological conditions on morphological variations of the green toad, Bufo viridis, in Turkey

Ten populations of the green toad, Bufo viridis, from three areas having different climatic regimes were compared. Multivariate analyses of morphological characteristics revealed three distinct populations of green toads: the first group inhabiting the lowland and humid localities was characterized by a small body size, the second group occurred at higher altitudes and in desert areas had a significantly larger body size, while the third group from the Mediterranean localities was characterized by a large tympanum, a longer tarsus and short first toes. Interestingly, the specimens from Giresun Island were clustered with the second group with a similar pattern of body size. Differences in the morphological characteristics among populations were positively associated with ecological conditions, and not with geographic distance. Some characteristics previously used for taxonomic purposes in B. viridis were shown to be associated with local climatic conditions.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo

Summary An investigation regarding the question of whether there exists a macula densa as part of the juxtaglomerular apparatus in the kidney of amphibians has been carried out. With the aid of a histochemical reaction for glucose-6-phosphate dehydrogenase activity, the presence of a macula densa zone as a specialized part of the distal tubule in the toad Bufo bufo was demonstrated. The functional significance of the high glucose-6-phosphate dehydrogenase activity in the macula densa cells is discussed.     

Vascularization of the pars intermedia of the hypophysis in the toad,Bufo bufo (L.) (Amphibia,Anura)

The vascularization of the pars intermedia of the hypophysis of the toad, Bufo bufo (L.) was studied by injection of a mixture of India-ink and gelatine into the circulatory system of the head via the arteria carotis communis. Further methyl-methacrylate corrosion casts of brains were made and the hypophysial region of the corrosion casts was examined with the scanning electron microscope. The results showed that the vascularization of the pars intermedia of the toad hypophysis consists of a single-layered vascular network, which is located on the ventral surface of the pars intermedia. The network is formed by capillaries, which primarily run caudally in a fan-like manner and which show only a few cross-connections. In the rostral region of the pars intermedia this network lies rather superficially, while in the caudal region it slightly penetrates the parenchyma. The vascular network originates from vessels of the neural stalk and from wide capillaries of the rostro-ventral region of the neurointermediate junction. The venous drainage of the pars intermedia is exerted by veins, which leave the caudal region and drain into the veins leaving the venous pole of the pars distalis. The flat, wide meshed vascular net on the ventral side of the pars intermedia, demonstrated in this study, fits into the concept that the pars intermedia of the anuran hypophysis is under the control of nerve fibers coming from the hypothalamus.