Axon-like processes in type III cells of taste organs
Article first published online: 3 FEB 2006
Copyright © 2006 Wiley-Liss, Inc.
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology
Volume 288A, Issue 3, pages 276–279, March 2006
How to Cite
Sbarbati, A., Merigo, F., Benati, D., Bernardi, P., Tizzano, M., Fabene, P. F., Crescimanno, C. and Osculati, F. (2006), Axon-like processes in type III cells of taste organs. Anat. Rec., 288A: 276–279. doi: 10.1002/ar.a.20313
- Issue published online: 20 FEB 2006
- Article first published online: 3 FEB 2006
- Manuscript Accepted: 24 NOV 2005
- Manuscript Received: 22 NOV 2005
- chemical senses;
- gustatory organs;
- epitheliomesenchymal interactions;
Type III cells of the taste organs are widely considered to be chemoreceptors. The present study was performed on the frog taste disk and describes an axon-like process in type III cells, which often contains a bundle of densely-packed parallel microfilaments. These processes pass through the basal membrane of the gustatory epithelium, running into the lamina propria (transbasal membrane processes, tBMPs). In their intraepithelial tract, tBMPs contain dense-cored vesicles revealing their origin from type III cells. Type III cells showing both classic nonrigid processes (with vesicles and nerve contacts) and tBMPs are present. The connective tract of a tBMP usually contains dense-cored vesicles only in its proximal portion. In some cases, the connective tract of tBMPs is almost perpendicular to the basal lamina. In other cases, it runs parallel to and below the basal lamina. Some tBMPs contact nerve fibers running in the subepithelial connective tissue; the contact area is rather wide but evident synapse-like junctions were never detected. Contacts between tBMPs and nerve fibers innervating basal cells are also found. In conclusion, the data demonstrate the existence of epithelial cells resembling primitive neurons that display an apical dendrite and axon-like basal processes. Until now, it was not considered possible that epithelial receptor cells extend processes out of the epithelium. © 2006 Wiley-Liss, Inc.
In vertebrates, information from external chemicals is obtained through two types of receptor cells. Olfactory and vomeronasal receptors are primary sensory neurons that emit an axon, which exits the epithelium and reaches the central nervous system. Taste receptor cells and solitary chemoreceptor cells are epithelial elements of bipolar profile, which transmit information to nerves through cytoneural junctions. The processes emitted from such cells do not exit the epithelium. The present study demonstrates that such a universally accepted dichotomy is not correct. There is clear evidence that epithelial taste receptor cells may behave like neurons, emitting axon-flike extensions that exit the epithelium. On the basis of their overall characteristics, these cells can be classified as type III taste cells.
The type III cells of the taste organs (Murray, 1986) are characterized by an apical process that reaches the free surface and by several basal processes. These latter contain dense-cored vesicles and contact axons (Royer and Kinnamon, 1991; Yee et al., 2001). Type III cells of taste organs have been described in all the vertebrate species that have been studied and are widely considered to be chemoreceptors. Often, they are simply called taste (or gustatory) cells. Due to their importance in gustatory processes, their morphology has been described in various studies, but some doubts remain about their neurochemistry and innervation.
Several studies have described the frog taste disk (FTD), which displays a very large diameter, facilitating physiological studies (for a detailed description of the organ, see also Osculati and Sbarbati, 1995). In addition, this model has recently been chosen for studies using multiphoton microscopy (Li and Lindemann, 2003). An FTD contains a large number of type III cells (600–900), which allows easy analysis of more elements than are found in small mammalian buds. The use of an amphibian model is also justified because type III cells, despite interspecies differences, display rather constant characteristics in all the species studied. The present study describes the morphology of an axon-like basal process of type III cells, which was obtained by serial sectional analysis of the FTD. The ultrastructural characteristics of this process are discussed in relation to their possible functions.
MATERIALS AND METHODS
Live wild frogs (Rana esculenta) of both sexes were obtained from a local supplier in summer and used for all experiments within 2 weeks of capture. The animals were anesthetized by immersion in 0.1% MS 222 (3-aminobenzoic acid ethyl ester; Sandoz) and were then decapitated. The tongue was immediately excised and cut into fragments with a razor blade. The fragments were fixed in 2.5% glutaraldehyde in Sorensen buffer for 2 hr, postfixed in 1% osmium tetroxide for 1 hr, dehydrated in graded ethanols, embedded in epon-araldite, and cut with an Ultracut E (Reichert, Vienna, Austria). The semithin sections were stained with toluidine blue. The ultrathin sections were stained with lead citrate and uranyl acetate and observed using an EM10 electron microscope (Zeiss, Oberkocken, Germany).
In the FTD, type III cells (Fig. 1A) are bipolar elements with cell bodies located in the intermediate layer. The single apical process is about 0.8 μm in diameter and is filled with microtubules and bundles of actin-like filaments. At its end (Fig. 1B), the apical process shows a cylindrical cytoplasmic extension similar to a large microvillus (diameter, ca. 0.6–0.8 μm; height, ca. 1 μm) with a smooth surface covered with a thick glycocalyx. A bundle of microfilaments completely fills the cylinder. They have a regular crystalloid arrangement and are inserted directly into the apical plasmalemma. The cell body has a high nuclear/cytoplasmic ratio and the nucleus usually shows a single indentation. There are few cytoplasmic organelles: a small Golgi complex, mitochondria, isolated dense granules, and glycogen particles.
Type III cells usually have several basal processes stemming directly from the cell body and showing an irregular course. The basal processes (Fig. 1A) are rich in actin-like filaments and microtubules are rarely visible. The basal processes reveal specific contacts with axons and numerous cytoplasmic dense-cored granules (Fig. 1A, inset). The cytoneural junctions of type III cells are not true synapses: the pre- and postsynaptic membranes usually run parallel, but there is no thickening. The cytoplasm of the axons contains glycogen, small mitochondria, and clear vesicles. Some type III cells are characterized by basal processes containing bundles of densely packed, parallel actin-like filaments (Figs. 2 and 3). Some ramified transbasal membrane processes (tBMPs) are present (Fig. 2C and D). Using serial sections, we detected type III cells showing both classic intraepithelial processes (with granules and nerve contacts) and tBMPs stemming from the cell body (Fig. 2E and F).
In tBMPs, an intraepithelial and an intraconnective tract are regularly present (Fig. 3A). In their intraepithelial tract, tBMPs contain dense-cored granules with diameters of 60–80 nm, revealing their origin from type III cells (Figs. 2C and D and 3A, C, and D). The connective tract of a tBMP usually contains dense-cored vesicles (Fig. 3D) only in its proximal portion (near the basal lamina), which can show a small dilatation (Fig. 3A and C). In this portion, a delicate network of actin-like filaments is present (Fig. 3A and C). The distal portion is almost totally filled with a compact bundle of actin-like filaments and other organelles are virtually absent (Fig. 3B). The intraconnective tract usually shows a diameter of 0.4–0.5 μm and a length of 4–5 μm. The intraconnective tract of tBMPs shows a variable inclination with respect to the basal lamina. In some cases, it is almost perpendicular to the basal lamina (Fig. 2A and B). In other cases, it runs parallel to and is just below the basal lamina (Fig. 3A and E). Usually, the intraconnective tract is not wrapped by glial-like cells or by other elements (Fig. 2B).
Some tBMPs contact axons running in the subepithelial connective tissue, also called the intrapapillary space (Fig. 3A). The contact area is rather wide, but evident synaptic-like junctions are never detected. The cytoplasmic membranes of the tBMP and of the axon are parallel and separated by a space of about 20 nm in which focal densities are visible (Fig. 3B). Contacts between tBMPs and nerve fibers innervating basal cells are also found (Fig. 3A).
As for numerical density, we never found more than one tBMP in a single FTD section. However, a single tBMP was found in virtually all the serial sections that we performed. Considering the fact that only a small part of a taste organ is observed in serial sections (an FTD has a diameter of 100–150 μm and a thin section is about 60–70 nm thick, so about 1,500–2,000 sections would be necessary to observe the whole structure), it is possible to assume that tBMPs are present in virtually all FTDs.
For comparison, Figure 4 shows the microvillar spikes of a Merkel-like (type IV) cell located in the basal portion of the FTD. As evident from the image, these cytoplasmic extensions are smaller and more numerous than tBMPs. They stem from the cell body of a different cell type and are intraepithelial.
The presence of protrusions extending into the connective tissue is rather unusual in epithelial cells and has not previously been reported in taste organs. This absence of a previous description may be due to several factors. In a taste organ, the density of cells showing tBMPs is low, and it is possible that tBMPs are not found in all species. Where they are present, their detection in a small mammalian bud would be difficult and in thin sections they could be considered to be axons. It is not surprising that these structures were detected in a giant taste organ, such as the FTD, and only by using a time-consuming serial sectioning approach.
The tBMP can be considered a new type of cell organelle or, more in detail, it could be classified as a morphological differentiation of the basolateral membrane of a taste cell.
Its functional interpretation is difficult, but it seems clear that a tBMP is not a simple mechanical adherence to the connective bed. The type III cell is not a mobile element, so it is difficult to link the presence of tBMPs to cell motility. It is also difficult to consider the tBMP as a new type of epitheliomesenchymal interaction. Usually, epitheliomesenchymal interactions are based on anatomical contact between an epithelial and a connective cell and are usually linked to developmental events (Villaro et al., 1998). tBMPs lack contacts with connective cells and they were found in well-differentiated cells, so there are no reasons for regarding them as structures linked to a developmental event.
The morphology of a tBMP is in some ways reminiscent of the rigid microvillus located at the end of the apical process of type III cells (Sbarbati et al., 1993). This latter is also characterized by a rigid membrane and contains a bundle of packed microfilaments. In the frog, the microvillus is short, but in other species (i.e., fishes), it is rather long. The rigid appearance of the apical microvillus is due to the presence of transmembrane proteins linked to the detection of tastants (Sbarbati et al., 1993). The possibility that type III cells use tBMPs to analyze the chemical composition of the intrapapillary space is intriguing, but there is no evidence that this is the case. The penetration of chemicals into this space has been confirmed (Zancanaro et al., 1990) and has recently obtained definitive confirmation using multiphoton microscopy (Li and Lindemann, 2003). Using tBMPs, a subset of bipolar taste cells could monitor chemical gradients existing between the oral cavity and the intrapapillary space. The tBMP might be involved in the evaluation of the intrapapillary microenvironment. This could be relevant to understanding physiological events such as papillary water uptake, or conspicuous water entry into the papillae during osmotic phenomena (Rapuzzi et al., 1983), water taste (Sugawara et al., 1989), or intravascular taste (Bradley and Mistretta, 1971; Bradley, 1973). In all these events, a modification of the microenvironment in the connective tissue of the lamina propria is possible.
At the present level of knowledge, it is difficult to know if the broad areas of contact between tBMPs and nerve fibers have a functional role. However, the structure we have found seems to represent a cytoneural junction of new type. In addition, our data clearly demonstrate that a single axon can contact both a basal (Sbarbati et al., 1988) and a type III cell and that these contacts are located in the connective tissue. This contact could mediate a reciprocal relationship between basal and type III cells.
In conclusion, the description of a new type of intraconnective process changes our image of type III cells in taste organs. Our work demonstrates for the first time the existence of epithelial cells resembling primitive neurons, which display an apical dendrite and an axon-like basal process. Until now, the idea that epithelial receptor cells might extend processes out of the epithelium was not considered possible. It is interesting to note that these differentiated elements are found in the FTD, which, despite its epithelial origin, displays a complex connectivity with elements (i.e., the Merkel-like basal cells) that, because of their numerous synaptic contacts, could be considered true epithelial interneurons.
- 1971. Intravascular taste in rats as demonstrated by conditioned aversion to sodium saccharin. J Comp Physiol Psychol 75: 186–189. , .
- 1973. Electrophysiological investigations of intravascular taste using perfused rat tongue. Am J Physiol 224: 300–304. .
- 2003. Multi-photon microscopy of cell types in the viable taste disk of the frog. Cell Tissue Res 313: 11–27. , .
- 1995. The frog taste disc: a prototype of the vertebrate gustatory organ. Prog Neurobiol 46: 351–399. , .
- 1986. The mammalian taste bud type III cell: a critical analysis. J Ultrastruct Mol Struct Res 95: 175–188. .
- 1983. Paracoronal cavity system and papillary water uptake. Boll Soc It Biol Sper 59: 1917–1921. , , .
- 1991. HVEM serial-section analysis of rabbit foliate taste buds: I, type III cells and their synapses. J Comp Neurol 306: 49–72. , .
- 1988. The fine morphology of the basal cell in the frog's taste organ. J Submicrosc Cytol Pathol 20: 73–99. , , , , , .
- 1993. Freeze-fracture characterization of cell types at the surface of the taste organ of the frog, Rana esculenta. J Neurocytol 22: 118–127. , , , , , .
- 1989. Mechanism of the water response in frog gustation: possible significance of surface potential. Brain Res 486: 269–273. , , .
- 1998. Relationship between epithelial and connective tissues in the stomach of the frog Rana temporaria during metamorphosis: an ultrastructural study. Tissue Cells 30: 427–445. , , , , .
- 2001. “Type III” cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin. J Comp Neurol 440: 97–108. , , , , .
- 1990. The chemoreceptor surface of the taste disc in the frog, Rana esculenta: an ultrastructural study with lanthanum nitrate. Histochem J 22: 480–486. , , , , .