Mast cells in the amphibian brain during development

Abstract

This is the first descriptive study of ontogenesis and anatomical distribution of mast cells in the developing brain of three different amphibian species. In the toad and the green frog, mast cells are preferentially located in: (i) the meningeal lining (pia mater), (ii) the choroid plexuses, both anterior and posterior, and (iii) the neuropil, in close association with the epithelial cell lining of blood vessels. It is only in the perennially aquatic African clawed frog that mast cells never appear inside brain ventricles and within the neuropil. Mast cells first become identifiable in brain of different species in different stages of development. While there are differences in the number of mast cells in different species at different stages of development, the number nearly doubles in all three species during the transition from pro-metamorphic stage of larval development to the peak of metamorphic climax. Furthermore, the number of mast cells is comparatively higher in the toad and remarkably lower in the fully aquatic Xenopus laevis, in which species the first appearance of identifiable mast cells during larval development occurs much later than in equivalent stages of development of the toad and the green frog. The secretory nature of mast cells can be assumed by the presence of cytoplasmic granules, which may show species-specific texture. Further experimental analyses are required to unveil the usefulness of mast cells in the amphibian brain.

Introduction

Following their description as ‘mast-zellen’ (cells packed with metachromatic granules) as early as 1879 (Ehrlich) in the human connective tissue, mast cells were identified also in lower vertebrates, including amphibians (Michels, 1923). These cells were not given much credit as to their role until the late second half of last century, when they were unequivocally described as one of the multi-potent cell types capable of secreting a multitude of chemical mediators such as cytokines, histamine, heparin, chemokines, proteases, neuropeptides and nitric oxide (Galli, 1990; Metcalfe et al. 1997). Mast cells derive from the hematopoietic stem cells and they became ‘famous’ for their immunoregulatory role(s) in humans (Marone, 1995). Indeed, among mammals, including humans, the most accredited role of mast cells is their involvement in acute inflammatory response against invading bacteria, helminthic parasites and harmless allergens (Galli & Wershil, 1996). The vast spectrum of roles now ascribed to mast cells has become a trigger to identify their presence in various body tissues, including the brain. Mast cells not only manifest distributive heterogeneity (in animal groups and in organs and tissues within the same group of animals) but also differences in their morphological features. Within the vertebrate taxa, they are highly abundant in mammals, comparatively less in birds and amphibians and, most notably, they may be totally absent in some species of teleost fish.

The intracranial distribution of mast cells (in the dura mater, leptomeninges and choroid plexus) has been described in mammals and birds, in which they have been reported to mediate in the permeability of the blood–brain barrier as well as in neuronal activation and/or suppression (Theoharides, 1990; Silver et al. 1996; Zhuang et al. 1996). Within the central nervous system, mast cells have been described in the parenchyma (mainly in the hypothalamus) with a predominantly perivascular location.

In amphibians, two morphologically distinct subtypes of mast cells exist containing either safraninophilic granules or alcianophilic granules (Minucci et al. 1997; Chieffi Baccari et al. 1998). Of these, only the presence of the former morphological subtype has been confirmed in the central (Chieffi Baccari et al. 2009) and peripheral nervous systems (Chieffi Baccari et al. 2000; Esposito et al. 2002). Obviously, their mere presence in the brain has posed questions related to their morphology and function. In addition, questions pertaining to where brain mast cells derive from, how they cross the blood–brain barrier and what factors influence (regulate) their proliferation and maturation are still subject to debate and further examination. A careful analysis of the literature shows that there is no information on developmental appearance, anatomical distribution, migration or function of mast cells in the central nervous system of amphibians. Consequently, it is imperative to construct a neuroanatomical picture of mast cells in the amphibian brain during development as well as in adulthood to provide a morphological setting to understand their putative role in the nervous system. Additionally, the multiplicity of their body distribution and frequency and role has been raising new questions, such as the relationship between mast cells and the environment of the animal. We have therefore examined three species of anuran amphibia: Xenopus laevis (a fully aquatic frog), Bufo bufo (a prevalently terrestrial toad; it goes to water only to reproduce), and Rana esculenta (a frog which lives in aquatic and terrestrial environments).

Materials and methods

In compliance with international guidelines, pre-metamorphic, pro-metamorphic and metamorphic climax tadpoles, as well as post-climax froglets/toadlets, were collected in the vicinity of Naples (Italy). At least three individuals (maximum five) were used for each selected stage, and the developmental stages were classified according to Nieukoop & Faber (1956) for X. laevis, Cambar & Gipouloux (1956) for B. bufo and Witschi (1956) for R. esculenta.

All animals were anaesthetized with tricaine methanesulfonate (MS 222, Sigma). Entire heads from pre-metamorphic tadpoles and brains from metamorphic climax and post-climax samples were rapidly dissected and immersed in Bouin's fluid. Paraffin-embedded, 7-μm-thick serial transverse sections were stained either with toluidine blue in Walpole buffer (pH 4.2) or with Alcian blue/safranin (AB/safranin): Alcian blue in 3% acetic acid (pH 2.2) and 0.5% safranin in HCl (pH 1.3). Histochemical conditions used in this study were based on prior work in which alternative fixatives and different pHs of toluidine blue and AB/safranin stainings were investigated (Chieffi Baccari et al. 1998). Whereas toluidine blue stains all sulphated glucosaminoglycans, AB/safranin method allows low sulphated-glucosaminoglycans (blue) to be distinguished from highly sulphated substances such as heparin (red). Slides were examined with a Leica DMLB microscope equipped with appropriate filter sets and a Canon Power Shot S50 digital camera. Images projected on a Sony screen were exported as TIFF files. Digital images were adjusted for brightness and contrast using Adobe Photoshop 8. Plate photomontage and lettering were done using CorelDraw 9. All sections from each of the three brains per stage of development were used for mast cell counting. These were expressed as number per section simply by dividing the total number of cells counted by the total number of sections for each brain. Nomenclature of brain areas was based largely on Ten Donkelaar (1998) and Pinelli et al. (1999).

Statistical analysis was carried out using Student's t-test for between-group comparisons and the levels between P < 0.01 and P < 0.05 were considered significant. All data were expressed as mean ± SD.

We chose to perform mast cell electron microscopy on choroid plexuses from metamorphic climax tadpoles essentially for two reasons: (i) the number of mast cells reaches a peak at this stage of development, and (ii) mast cells appear more concentrated in this tissue. Choroid plexus was also collected from pro-metamorphic tadpoles of the toad because of a comparatively greater abundance than in either species of frog. Each sample was promptly immersed in Karnovsky buffer (pH 7.4) and postfixed in Millonig's phosphate-buffered 1% osmium tetroxide. Ultrathin sections were stained with 4% uranyl acetate followed by 1% lead citrate.

Results

Ontogenesis and anatomical distribution of mast cells

Bufo bufo

At stage IV₇ (forelimb buds visible; early pre-metamorphosis), a few immature type mast cells were observed in the telencephalon, diencephalon, mesencephalon and rhombencephalon. These spherical cells (diameter about 6 ± 1 μm) were orthochromatic with toluidine blue and contained very little secretory material (Fig. 1a,b). Such cells were particularly abundant in the ventrolateral and ventromedial pia mater at the level of the olfactory bulbs (Fig. 1a). Scattered roundish mast cells were seen in the infundibular recess (third ventricle) adjoining the ventricular lining and sometimes along the pial layer of the optic roof (Fig. 1b).

Fig. 1.

Bufo bufo brain sections during development. (A) Stage IV₇: enlargement of boxed area in the inset showing round, immature type, mast cells (arrows) in the pia mater along the ventral surface of the olfactory lobes. (B) Stage IV₇: a mast cell along the pial layer of the optic roof (arrow). (C) Stage IV₉: a mast cell in the third ventricle near the habenula. (D) Enlargement of a mast cell arrowed in (C). (E) Stage IV₉: a mast cell near blood capillaries in the posterior dorso-lateral rhombencephalon. (F) Stage IV_11/12: mast cells (arrows) in the pia mater along the medial septal area. (G–H) Stage IV_11/12: mast cells (arrows) along the upper lateral surface of diencephalon. Note the bilateral symmetry of their distribution. (I) Stage IV_14/15: elongated mast cells in the pia mater (arrows) surrounding the olfactory-vomeronasal nerves complex (asterisk) proximal to the olfactory bulbs. (J) Same stage as in g: arrow points to elongated mast cells in the medial septal area (cf. Fig. 1f). (K) Stage IV₁₇ with arrows indicating metachromatic elongated mast cells near the habenula (asterisk). (L) Same stage as k with elongated mast cells (arrows) in the pia mater between the cerebellum (asterisk in the inset) and rostral rhombencephalon. The boxed area of the inset has been magnified for each figure. III, third ventricle; IV, fourth ventricle; LV, lateral ventricle; OV, optic ventricle. Scale bars: (A–C, E–L) 50 μm, (D) 10 μm, (insets) 100 μm.

At stage IV₉ (pre-metamorphosis), the anatomical distribution and frequency of mast cells remained grossly unaltered compared to the earlier age group, and they appeared orthochromatic with toluidine blue. Strongly orthochromatic mast cells were observed in the third ventricle, flattened against the ventricular wall (Fig. 1c,d). Prominent, roundish mast cells were also observed in the dorsolateral parenchyma of the posterior rhombencephalon, closely associated with blood capillaries invading this brain area (Fig. 1e). In general, in these tadpoles, mast cells were also located in the hypothalamic area, along the ependymal lining and in the innermost layer of the meninges surrounding the brain, the pia mater.

In stages IV_11/12 tadpoles, mast cells were comparatively more numerous in the brain than in the previous stage of development (Fig. 2); they were still predominantly orthochromatic with toluidine blue (Fig. 1f–h) and prevalently Alcian blue-positive with the AB/safranin sequential reaction (not shown). In this stage group, furthermore, mast cells were also observed in the telencephalon, in the pia mater around the medial septum (Fig 1f). Some mast cells were observed in the epithelial cell lining of small blood vessels within the brain parenchyma. In the diencephalon, mast cells were found distributed dorsally and dorso-laterally in the proximity of habenulae, some associated with pia mater (Fig. 1g,h) and some within the third ventricle adjacent to its ependymal lining. The distribution of mast cells in this area shows an easily discernible bilateral pattern. In these tadpoles, there were fewer mast cells in the mesencephalon and rhombencephalon.

Fig. 2.

Graphical representation of mast cell number (MCN) per section at various stages of development in the brain of Bufo bufo, Rana esculenta and Xenopus laevis. In all three species, the number of mast cells increases progressively during development, reaching the maximum at metamorphic climax. At this stage, MCN per section is comparatively higher in B. bufo than in R. esculenta or X. laevis. * P < 0.05, IV_11/13 and IV₁₅ vs. IV₇ and IV₉ (B. bufo); 26/28 vs. 22/23 (R. esculenta); 32/33 vs. 29/30 (R. esculenta); **P>0.01, IV₁₇ vs. IV₇ and IV₉ (B. bufo); 32/33 vs. 22/23 (R. esculenta); 63/66 vs. 51/55 and 57/58 (X. laevis).

In the brain of stage IV_14/15 tadpoles, mast cells were mostly elongated in shape and were metachromatic with toluidine blue, which stains highly sulphated substances such as heparin (red) (Fig. 1i,j), and safranin-positive with AB/safranin staining (not shown). Rostrally, many elongated mast cells were observed around the olfactory-vomeronasal nerve complex ventral to the olfactory bulbs (Fig. 1i). Mast cells in the telencephalon were distributed along the medial septum (Fig. 1j) and some of them were closely associated with blood vessels penetrating the brain parenchyma. Although rare in lateral ventricles, mast cells were always associated with the ependymal lining and many were distributed in the pia mater. In the diencephalon, some mast cells were located in the epithelial lining of the anterior choroid plexus, whereas some others were present not only within the neuropil (associated with small blood vessels) but also attached to the ependymal lining of the third ventricle. In the mesencephalon, mast cells were associated with pia mater. Mature and elongated mast cells were present along the ependymal lining of dorsal infundibulum. Some cells were located at the level of the rhombencephalon, closely associated with the blood capillary bundle of the posterior choroid plexus.

In newly metamorphosed toadlets (developmental stage IV₁₇), brain mast cells appeared as mature cells; they were elongated, with a diameter of about 15 ± 3 μm (nuclear diameter 5 ± 1), and strongly metachromatic (Fig. 1k,l). They were distributed mainly in the telencephalon, along the medial septum, and in the diencephalon, where they were particularly abundant near the habenulae (Fig. 1k). Several elongated mast cells were observed in the proximity of cerebellum. Here, they were precisely located in the meningeal layer lying between the cerebellum and the rhombencephalon (Fig. 1l).

During development, the number of mast cells per brain section increased significantly (P < 0.05) at stages IV_11/13 (2.8 ± 0.3 mast cells per section) and IV₁₅ (3.2 ± 0.5 mast cells per section) compared to the earlier stages of development (1.3 ± 0.1 mast cells per section at stage IV₇ and 1.5 ± 0.1 mast cells per section at stage IV₉) (Fig. 2). The number of mast cells in the brain increased progressively and peaked at the metamorphic climax when tail resorption is completed. The mast cells number at this stage (5.3 ± 0.9 mast cells per section) is significantly (P < 0.01) higher with respect to stages IV₇ and IV₉ (Fig. 2).

Rana esculenta

In stage 22/23 tadpoles, only a few mast cells were observed in the brain. They were generally roundish (diameter of about 5 ± 1 μm) in appearance and weakly metachromatic with toluidine blue (Fig. 3d) and prevalently Alcian blue-positive with AB/safranin staining (not shown). Proceeding antero-posteriorly, occasional mast cells were observed in the telencephalon, associated with blood vessels of the brain parenchyma as well as within the lateral ventricles. In this site they appeared strongly orthochromatic (Fig. 3a,b) and were generally flattened against ventricular lining. In the diencephalons, only occasionally was a mast cell or two found along the ependymal lining of third ventricle and on the brain side of the blood capillaries within the anterior choroid plexus (Fig. 3c,d). Caudally, in the rhombencephalon, some mast cells were observed within the fourth ventricle.

Fig. 3.

Rana esculenta brain sections during development. (A–B) Stage 23: enlargement of boxed areas in the inset showing mast cells in the lateral ventricles (arrows). (C) Same stage as (B). A mast cell adjacent to the anterior choroid plexus (upper arrow) and another attached to the ventricular (III ventricle) lining (lower arrow). (D) Enlargement of the lower mast cell in (C). (E) Stage 24/25 tadpole showing numerous mast cells in the anterior choroid plexus (arrows). (F) Stage 27 with a mast cell (arrow) in the optic ventricles. (G) Stage 30 with elongated metachromatic mast cells (arrows) in the richly vascularized pia mater (arrow head) at the level of medial septum. (H) Early metamorphic climax with metachromatic elongated mast cells (arrows) in the same area as in (G). (I) Mast cells (arrows), filled with granules, in the anterior choroid plexus of a newly metamorphosed froglet (stage 33). (J) Same stage and area as (I) showing magnified mast cells. (K) Same stage as (I) showing elongated mast cells (arrows) in the fourth ventricle (IV in the inset) extension in between cerebellum (upper) and rostral rhombencephalon. (L) Same stage as (I) showing roundish metachromatic mast cells (arrows) in the posterior choroid plexus. III, third ventricle; IV, fourth ventricle; LV, lateral ventricle. Scale bars: (A–C, E–I, K, L) 50 μm, (J) 25 μm, (D) 10 μm, (insets) 100 μm.

At stage 24/25 (early pre-metamorphosis), mast cells were still round in appearance and weakly metachromatic. In the telencephalon, mast cells were observed in lateral ventricles, with a sort of bilateral symmetry in their distribution. In the diencephalon, mast cells were present along the ependymal lining of the third ventricle, some near the habenulae, and many were located dorsally in association with the epithelial lining of the blood capillaries of the anterior choroid plexus (Fig. 3e). Similar to the earlier stage of tadpoles, some mast cells were observed along the lining of the fourth ventricle and some were observed associated with the blood capillary bundle of the posterior choroid plexus. Occasionally, mast cells were observed also ventro-medially in the meningeal lining of the rhombencephalon.

In the brain of stage 27 tadpoles (advanced pre-metamorphosis), all mast cells appeared mature and metachromatic with toluidine blue. They were either oval or elongated in shape. In the telencephalon, mast cells were observed in lateral ventricles. Numerous mast cells were present in the third ventricle, throughout its length from the diencephalon to infundibulum, with a relatively higher frequency in the habenular region, where they were closely associated with the anterior choroid plexus. At the level of the diencephalon, in fact, mast cells were observed in the central part of third ventricle and in the infundibular recess. Interestingly, in this stage group, some orthochromatic mast cells were also observed in the mesencephalic ventricle (optic ventricle) (Fig. 3f). In the rhombencephalon, some such cells were present in the fourth ventricle, some others were in the cerebellum and still others were observed within the posterior choroid plexus.

Also in the stage 29/30 group (pro-metamorphosis), some mast cells were metachromatic with toluidine blue (Fig. 3g) and many of them were Alcian blue-positive with AB/safranin reaction (Fig. 4a). Some mast cells were oval in shape, whereas others had an elongated body. Mast cells in the telencephalon were numerous along the peritoneal lining of the medial septal area (Fig. 3g) and were frequently observed in lateral ventricles as well as the diencephalon. Often, many such cells were observed in the infundibular recess, adjacent to its ependymal lining and neighbouring blood vessels. Further caudal, some mast cells were present in the mesencephalon and rhombencephalon, mainly along the ventricular lining and in the posterior choroid plexus.

Fig. 4.

Paraffin sections from brain of Rana esculenta tadpoles. (A) Mast cell (arrow) of a tadpole at stage 29 showing AB-positive and weakly safranin-positive granules. (B) Elongated mast cell of a metamorphic climax sample strongly staining in red with AB/safranin reaction. Scale bars: 8 μm.

In early metamorphic climax the increased frequency of elongated and metachromatic mast cells in the medial septal area was remarkable (Fig. 3h).

At stage 32/33 (advanced metamorphic climax – newly metamorphosed froglets), the brain contained a remarkable number of strongly metachromatic big-sized mast cells in close association with the epithelial lining of the anterior choroid plexus (Fig. 3i). At this stage of development, mast cells appeared more mature than those observed in the earlier stages of development, and were often considerably bigger in size (diameter of about 15 ± 2 μm; nuclear diameter 5 ± 1), with an elongated shape; the cells showed an intense metachromasia with toluidine blue (Fig. 3j) and were strongly safranin-positive (Fig. 4b). Mast cells were abundant in the telencephalon. Here, they were distributed mainly along the medial septal meningeal lining, some within lateral ventricles and others in close association with blood vessels. Many mast cells were observed in the diencephalon region, associated with the anterior choroid plexus, ventricular ependymal lining in the infundibular recess as well as ventrally in the pia mater surrounding the brain. A few mast cells were observed in the mesencephalon, within the optic ventricles. Strongly metachromatic and slightly elongated mast cells were observed at the level of the cerebellum (Fig. 3k), in the pia mater. In the rhombencephalon, other mast cells were present along the ependymal lining of the fourth ventricle, in blood vessels entering the brain tissue and in the posterior choroid plexus (Fig. 3l).

Mast cell number increased from 0.05 ± 0.01 mast cells per section in stage group 22/23 to 0.25 ± 0.04 mast cells per section in stage group 26/28 (Fig. 2). Mast cell number at stage 32/33 (1.8 ± 0.4 mast cells per section) increased significantly (P < 0.01 and P < 0.05, respectively) compared to that in stages 22/23 (0.25 ± 0.04) and 29/30 (0.6 ± 0.1) (Fig. 2).

Xenopus laevis

In X. laevis, the first roundish mast cells (diameter of about 4 ± 1 μm), orthochromatic with toluidine blue (Fig. 5b) and AB positive with AB/safranin reaction (not shown), were observed in the forebrain pia lining of one sample of stage 37/38 tadpole (hind limb buds not visible) (Fig 5a,b).

Fig. 5.

Xenopus laevis during development. (A) Stage 37 tadpole with a rarely seen round orthochromatic mast cell (arrow) in the pia mater of forebrain. (B) Enlargement of the mast cell pointed in a. (C) Stage 55 with a roundish mast cell (arrow) in the meningeal layer of the olfactory bulbs. (D) Stage 58 with a mast cell (arrw) in the pia mater near the medial septum. (E) Enlargement of the mast cell pointed in d. (F) Stage 58 with mast cells (arrows) in the pia mater along the dorso-lateral area of the rostral rhombencephalon. (g–h) Stage 66 (newly metamorphosed froglet) brain containing mast cells in the dorsal medial septal area (G) in the vicinity of blood vessels (arrow heads) and in medioventral pia mater (H). (I) Stage 66 with mast cells in the meningeal lining of the infundibulum. Asterisk shows a blood vessel. (J–K) Mast cells (arrows) in the pia mater around the anterior rhombencephalon. (L) An elongated mast cell (arrow) situated at the base of a cranial nerve sprouting from the brain at mid-rhombencephalon. III, third ventricle; IV, fourth ventricle; LV, lateral ventricle. Scale bars a, c, d, f–l = 25 μm; b, e = 10 μm; insets = 100 μm.

Subsequently, much later in development (stage 51/55, pre-metamorphosis), mast cells were observed in all brains examined. In the telencephalon, mast cells were metachromatic with toluidine blue and roundish or oval in shape; some such cells were always associated with the olfactory bulbs (Fig. 5c). In the diencephalon, a few mast cells were present in the dorsal region, closely associated with epiphysis and precisely located in the meningeal lining.

At stage 57/58 of development (advanced pre-metamorphosis), the brain mast cells appeared metachromatic (Fig. 5e) and weakly safranin-positive, although a few were rich in granules and showed a rather roundish morphology. A few mast cells were found in the telencephalon, along the median septum (Fig. 5d,e). In the diencephalon, some mast cells were located dorsally near the habenulae, and many such cells were observed within the anterior choroid plexus on its epithelial lining. Mast cells were more numerous in the lateral peripheral area of infundibulum, located in the neighbourhood of large blood vessels that run laterally and dorsally to it. At this stage, only a few mast cells were observed in the mesencephalon, laterally and ventrally, and in the rhombencephalon, where they were located in the pia mater dorso-lateral to the hind brain and were of a roundish-looking immature type (Fig. 5f).

At stage 63/66 of development (metamorphic climax – newly metamorphosed froglets), brain mast cells were appreciable in size (diameter of about 8 ± 2 μm; nuclear diameter 4 ± 1) but the majority still showed a roundish body. They were metachromatic (Fig. 5g–l) and safranin-positive. Mast cells were almost always observed in close association with cranial nerves, meninges (pia mater) or nearby blood vessels. Several mast cells were present in the telencephalon, particularly along the dorsal and ventral extension of the medial septum, where they were often associated with particularly voluminous blood vessels (Fig. 5g,h). In the diencephalon, some mast cells were located ventrally, in the preoptic recess, and dorsally at the level of the habenulae, in the anterior choroid plexus. Mast cells were present also in the hypothalamic area, especially at the level of the sub-commissural organ; here these cells appeared associated with the meningeal lining and nearby large vessels running laterally to the infundibulum (Fig. 5i). At the level of mesencephalon, different mast cells were present both dorsally and laterally. In the rhombencephalon, they were distributed both basally and laterally, associated with blood vessels entering the brain tissue (Fig. 5j, k), and not the blood vessels of the posterior choroid plexus. Some elongated metachromatic mast cells were also observed in association with rhombencephalon nerves or along their course (Fig. 5l).

At stages 63/66 mast cell number was significantly (P < 0.01) higher (0.6 ± 0.1 mast cells per section) than stages 51/55 and 57/58 (0.02 ± 0.006 mast cells per section and 0.04 ± 0.01 mast cells per section, respectively) (Fig. 2).

Ultrastructure

The immature mast cells in B. bufo at stage IV₁₅ of larval development showed an elongated heterochromatic nucleus with peripheral condensations of chromatin, a few prevalently electron dense granules and numerous microvilli on cell surface (Fig. 6a).

Fig. 6.

Electron micrographs. (A) Immature mast cell from Bufo bufo at stage IV₁₅ of development showing a few electron dense secretory granules (arrow). Numerous microvilli are present on the cell surface. N, nucleus. (B) An elongated mast cell from B. bufo at metamorphic climax, showing the cytoplasm filled with numerous heterogeneous, polymorphic granules. Matrix of the granules, of granular appearance, contains lamellar inclusions. Occasionally, granules are strongly electrondense (arrows); arrowheads, microvilli. (C) An elongated mast cell from Rana esculenta at metamorphic climax, showing numerous electrondense cytoplasmic granules. The granules are heterogeneous in shape. (D) A round mast cell from Xenopus laevis at metamorphic climax, showing the cytoplasmic granules of different electrondensity. Numerous microvilli (arrows) are present on the outer surface. Scale bars a−d = 2 μm.

At the metamorphic climax, in B. bufo, mast cells were characterized by a predominantly heterochromatic nucleus and numerous secretory granules that limit the vision of other cellular structures and organelles (Fig. 6b). The surface of mast cells appeared rich in microvilli. The secretory granules were mostly round or ovoid in shape. The matrix of the majority of granules was of granular appearance and often showed lamellar or fusiform inclusions. Some granules were strongly osmiophilic (Fig. 6b).

The mature mast cells of R. esculenta contained numerous secretory granules, of variable shape (ovoid or round) and size (Fig. 6c). The granules were homogeneously osmiophilic and they did not show the substructural architecture typical of adult mast cells (Chieffi Baccari et al. 1998). The surface of frog mast cells was not adorned with surface folds (Fig. 6c).

In X. laevis, round mast cells showed membrane-bound cytoplasmic granules predominantly spherical in shape (Fig. 6d). The matrix of the majority of them appeared homogeneous and varied from weakly to strongly osmiophilic. The outer surface of mast cells was characterized by the presence of microvilli (Fig. 6d).

Discussion

This study represents the first attempt to describe developmental and quantitative changes in mast cell population in the amphibian brain during development. Attention is also focused on their morphological characteristics. This is a descriptive study and its scope was not discussion of either functional aspects or local brain factors influencing the population of mast cells in the amphibian brain. Suffice it to say that, in mammals, it is now recognized that mast cells originate from pluripotent stem cells of bone marrow, circulate in the blood as pro-mast cells and differentiate and mature in peripheral tissues, including brain, in response to local microenvironment (Tsai et al. 1991, 2000; Irani & Schwartz, 1994; Yong, 1997).

The only report about the embryonic origin of mast cells in non-mammalian vertebrates was carried out by Andrew & Rawdon (1987). These authors observed that connective mast cells originate from the mesoderm rather than the neural crest. Even less is known about the ontogeny of mast cells in the central nervous system. Among non-mammalian vertebrates, research on the ontogeny of brain mast cells has so far been attempted only in pigeon (Zhuang et al. 1999). Mast cells first appear in embryos at 13–14 days, in the pia mater; subsequently, 4–5 days after hatching, they appear in the telencephalon and later, at 3 weeks after hatching, in the medial habenula. The number of mast cells in the medial habenula increases during development, reaching a peak in birds coming to puberty and decreasing thereafter. In this area, mast cells were mature, showing metachromasia, Alcian-blue/safranin-positive granules and immunoreactivity to GnRH antibody. Hence in birds, mast cells enter the CNS as ‘immature’ cells and subsequently differentiate inside the neuropil, at least in the medial habenula.

More is known of the ontogeny of mast cells in the brain of mammals. In rats and mice, contrary to what has been described in birds, mast cells enter the brain as relatively ‘mature’ cells through the network of blood vessels (Lambracht-Hall et al. 1990). It should to be mentioned here that the blood–brain barrier of choroid plexuses does not isolate the brain from cellular elements of the immune system such as mast cells, which can reach the brain by crossing the blood capillary endothelial cells. Also, blood vessels penetrate the brain at numerous points but may remain separated from the neural tissue by a basal lamina. It is plausible to assume that at such points, the mast cells might invade the brain attached to the capillary wall. This is what that presumably happens in the amphibian brain as well. In fact, during development, cells with the colouring characteristics of immature mast cells appear in pia mater, the thin vascularized meningeal connective tissue lining surrounding the developing brain. At subsequent stages of larval development, mast cells appear and progressively increase in number around many blood capillaries within the brain tissue. In fact, as confirmed in mammals (Khalil et al. 2007), in the green frog and common toad the mast cells are localized in close association with the brain side of the cell lining of the blood vessels (these cells are in reality modified ependymal cells). In the developing toad or frog brain, it is only rarely that a mast cell can be observed in the neuropil away from a blood vessel. Thus, it seems that in the vertebrate brain, the number of mast cells in the brain may increase progressively during development (cf.Zhuang et al. 1999; Khalil et al. 2007; Michaloudi et al. 2007).

One of the special characteristics of mast cells is their extreme heterogeneity, not only interspecific but also intraspecific. The morphological and biochemical heterogeneity of mast cells between different anatomical locations, within an individual, seem to be determined by the local microenvironment that directs the function (see Khalil et al. 2007). The present study is part of a project on the diversity of mast cell population among species of frogs and toads. To understand whether the habitat influences the heterogeneity of mast cells (ontogeny, distribution and morphological characteristics) we have chosen three species of anuran amphibia with different habitats. In particular, we followed the ontogeny of brain mast cells until metamorphosis in X. laevis living perennially in water, R. esculenta with a partially aquatic life, and B. bufo with closely terrestrial habitats. The results of this study showed that, during ontogeny, in three species of amphibians, there are differences in appearance and maturation of mast cells, and while their distribution within the pia mater throughout the length of the brain is quite similar, their distribution within the brain differs somewhat. In fact, only in B. bufo and R. esculenta were the mast cells always observed within the brain, in a perivascular location on the brain side of the blood capillaries invading the neuropil and inside the brain ventricles flattened against the ependymal cell lining. One important point is that the highest number of mast cells appear in the brain at metamorphic climax in all three species. Indeed, in R. esculenta the remarkable increase in the number of mast cells during the metamorphic climax was described earlier in the tongue (Chieffi Baccari et al. 2003). This may lead to speculations of a functional nature but we here will limit ourselves in saying that mast cells may play important role(s) in a period (e.g. metamorphic climax) when the entire body of the animal is transforming. In the adult, nevertheless, in all three species the number of mast cells per section is relatively higher than at the time of metamorphic climax (data not shown).

In R. esculenta and B. bufo mast cells appear in an early stage of development (at stage 22 in R. esculenta, at stage IV₇ in B. bufo), whereas in X. laevis they appear only at stage 51/55. In all three species the number of mast cells increases with the progression of larval development, reaching a peak at metamorphic climax. At this stage, the number of mast cells per brain section is comparatively higher in B. bufo than in R. esculenta or X. laevis.

In all three species of amphibians, the mast cells first become detectable as immature cells (like in birds; Zhuang et al. 1999), round in shape, with few orthochromatic secretory granules, and are prevalently alcianophilic with AB/safranin sequential staining. The immature cells contain only a small amount of electron-dense secretory granules. During development, they differentiate gradually through to the metamorphic stage, becoming elongated and strongly metachromatic with toluidine blue and safranin-positive. At the ultrastructural level, their secretory granules exhibit species-specific substructure. Interestingly, it seems that in R. esculenta mast cells mature in an earlier stage of larval development than in the other two species. Hence, it will be interesting to study, during ontogeny in the three species of amphibians, the levels of growth factors (interleukins, stem cell factor, etc.) known to induce differentiation and maturation of mast cells. This could explain the differences in the time of their first appearance in the brain and maturation of mast cells in the three species.

We have not observed substantial differences in the distribution of mast cells in various areas of the brain of the three species. The mast cells are distributed in all areas of the brain and, in all three species their greater density was detected in the medial septum of the telencephalon and in the diencephalic area, associated with anterior choroid plexus.

Contrary to what has been described in the pigeon (Zhuang et al. 1997), mast cells have rarely been observed inside the neuropil. One interesting difference that we have observed in the distribution of mast cells in the brain between the aquatic species and the other two species is the absence, in X. laevis, of mast cells in the ventricles and adjacent to blood vessels in the brain parenchyma. In this species, in fact, brain mast cells are exclusively confined to the pia mater.

In addition to differences in the appearance, maturation and density during ontogeny, mast cells of the three species examined show secretory granules with a different sub-structure, and differently to Bufo and Xenopus, Rana brain mast cells did not show microvilli on the outer surface, as previously described in mast cells of some non-neural tissues in this species (Chieffi Baccari et al. 1998).

In conclusion, the results of this study indicate that the predominantly terrestrial species, B. bufo, shows the highest number of mast cells in all larval stages until metamorphosis. The overall number of mast cells per brain in the toad must be much higher than in either of the frog species. Simply counting the total number of 7-μm sections per brain (from the olfactory bulb up to the medulla), it appears that during development (from the early pre-metamorphic stage to the climax), Xenopus brain grows nearly five times in length (from roughly 0.7 mm to 3.35 mm), Rana brain nearly 2.5 times (from ∼ 2.7 to 6.1 mm), and Bufo brain nearly 1.5 times (from ∼ 2.1 to 3.0 mm). The overall number of mast cells per brain in Xenopus, Rana and Bufo may thus amount roughly to 4–290, 50–1600 and 400–2200, respectively, from the earliest pre-metamorphic tadpole to the froglet/toadlet stage. It has recently been ascertained recently in mammals (mainly land-dwelling species) that the mast cells have a role of defence in viral infections, such as HIV infection (Patella et al. 2000) and streptococcal infections (Di Nardo et al. 2008); in line with this, we are inclined to speculate that for the habitat they live in, the toads may need a higher number of brain mast cells compared to aquatic species. Indeed, the presence of a large number of mast cells in the brain of the crested newt (Mazzi, 1954), which spends a long period of its life on the mainland and returns to water only during reproduction, would point in this direction. Our observation of the lowest density of mast cells in the brain of the exclusively aquatic species, the African clawed frog (X. laevis), could be substantiated by some earlier data on the total absence or very low frequency of mast cells in non-nervous tissues of bony fishes (for review see Reite, 1998). However, some recent studies, again in teleosts, may be in contrast with this supposition; in fact several eosinophilic granule cells have been observed and have been considered the equivalent of mast cells (for review see Reite & Evensen, 2006). This observation has led to some debate. Assuming that mast cells (= eosinophilic granule cells) have a role in host defence, the challenge from pathogenic agents encountered by different species of teleosts in their habitat may be reflected in the pattern of mast cells and the nature of their role. Undoubtedly, further studies are required to understand whether environmental factors influence the differentiation and survival of mast cells in the brain.