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Ultrastructure of Merkel-like cells labeled with carbocyanine dye in the non-taste lingual epithelium of the axolotl
Neuroscience Letters 178 (1994) 14
ELSEVIER
NEUROSCItNCE IETItRS
Ultras ructure of Merkel-like cells labeled with carbocyanine dye in the non-taste lingual epithelium of the axolotl T a k a t o s h i N a g a i a'*, H i r o m i c h i K o y a m a b aDepartment of Physiology, Teikyo University School of Medicine, Tokyo 173, Japan bDepartment of Anatomy, School of Medicine, Yokohama City University, Yokohama, Japan
Received 14 June 1994; Revised version received20 June 1994; Accepted 28 June 1994
Abstract
Fluorescent carbocyanine dye (diI), applied to the glossopharyngeal (IX) nerve of the axolotl, transneuronally labeled solitary cells in the non-taste lingual epithelium. With diaminobenzidine (DAB), the diI was photoconverted to a dark, electron-dense product. The labeled cell had a large nucleus with invaginations, dense-cored vesicles in the cytoplasm, and finger-like processes. These are reminiscent of morphological features of cutaneous Merkel cells, suggesting that solitary cells innervated by the IX nerve are associated with mechanosensory function of the IX nerve system. Key words." Mechanotransduction; Merkel cell; Salamander; Lingual epithelium; Fluorescent dyes; Photoconversion; Transmission electron microscopy
The glossopharyngeal (IX) nerve of the amphibian responds to chemical and mechanical stimuli applied to the lingual epithelium [5,9,11,15]. Chemical stimuli are transduced by taste receptor cells in the taste bud, whereas trangduction elements for mechanical stimuli remain unclear. One of the possible elements for mechanical transduction in the IX nerve responses is the basal cell in the taste bud, because the cell has cytological features similar to cutaneous Merkel cells [1,3,13,14] and is innervated by the IX nerve [8], although the function of the Merkel cell is controversial [2]. We recently examined the innervation pattern in the lingual epithelium of axolotls using transneuronal labeling with fluorescent carbocyanine dye, and found that the IX nerve innervates not only cells in the taste buds but also solitary cells in the non-taste lingual epithelium [8]. However, this study did not reveal cytological features of the solitary cells. Therefore, in the present study, fluorescence of solitary cells was photoconverted [7,10] into an electron-
*Corresponding author. Fax: (81) 3-5248-1415. 0304-3940194l$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved SSDI 0304-3940(94)00511-7
dense reaction product for subsequent electron microscopic observations. Juvenile axolotls, Ambystoma mexicanum, of the white strain were obtained from the Indiana University Axolotl Colony and raised in our laboratory. Three axolotls of about 7 month old (10 cm total length) were anesthetized in 0.2% MS222 (tricaine methanesulfonate; Sankyo) for 20-30 min and were perfused transcardially with a fixative containing 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). Glutaraldehyde level was kept low to avoid substantial background fluorescence. The tongues were dissected out, kept in the same fixative overnight, and transferred to the fixative without glutaraldehyde. A 2.5% solution ofdiI (Molecular Probes, Eugene, OR, U.S.A.) dissolved in 50% ethanol and 50% dimethyl sulfoxide was applied to the IX nerve stump. Methods of application of diI were described in detail elsewhere [8]. After the application of diI, the tongue was covered with 5% agar to prevent the accidental spread of the dye to the surrounding tissues and placed in the paraformaldehyde solution in a 37°C oven for 2 months. The tissue was then freed from the agar, reembedded in gelatin, hardened overnight in the same fixative and sectioned on a Microslicer
2
Z Nagai, H. Koyama/Neuroscience Letters 17~; (1994) 1 4
Fig. I. Photomicrographs of the dorsal surface of wholemounted lingual epithelium. A: fluorescent dil-labeled solitary cell (arrow) and taste buds (arrow heads) in the epithelium. The upper solitary cell in A is out of focus in B. B: photoconverted epithelium with dark end-product. Note corresponding solitary cell (arrow) and taste buds (arrowheads) in A and B. Bar = 100/Jm.
40 nm) were examined, usually without staining, at 75 kV in a Hitachi H-600 transmission electron microscope. If necessary, the thin sections were stained with uranyl acetate and lead citrate. DiI intensely labeled the nerve fibers beneath the lingual epithelium, as well as many taste buds and solitary cells which were distributed in the lingual epithelium (Fig. 1A). Labeled cells outside of the taste buds (nontaste epithelium) were easily located with fluorescent microscope, since labelings in the non-taste epithelium usually occurred in a solitary cell, not in a cell cluster like a taste bud [8]. In the presence of DAB, fluorescent illumination produced dark-brown reaction product (Fig. I B). Brightly fluorescent cells in the non-taste epithelium were turned into the darkest (arrow in Fig. 1). Thus, small tissues containing a photoconverted cell could easily be excised for subsequent light and electron microscopies. Labeled solitary cells were usually found in the deeper layer of the non-taste epithelium (Fig. 2). In the labeled solitary cells, the photoconversion product ofdiI occurred as patches of electron dense plasma membrane and outer nuclear envelope (Fig. 3). A large nucleus with an invagination was surrounded by a limb of cytoplasm which contained many vesicles. The cell had some finger-like processes extending into the neighboring cells. In the lingual epithelium of axolotls, taste bud cells form a rosette-like arrangement distinct from general epithelial cells, whereas solitary cells in the epithelium do not [8]. Therefore, without some kinds of labeling, it is difficult to examine a tissue containing solitary cells histologically. The dark brown end-product of diI photoconversion allowed us to locate the solitary cells. Electron microscopy revealed that the labeled solitary
(Dosaka EM Co., Ltd., Kyoto). Thick sections (200/tm) of the tissue were cut in the tangential plane to the dorsal surface of the tongue. Specimens were viewed by means of a Nikon fluorescence microscope equipped with an epi-illumination system (rhodamine filter set, G-2A). The sections were photoconverted with diaminobenzidine (DAB) to produce a dark, electron-dense product, following the procedure described by Sandell and Masland [10]. The sections were immersed in a solution of 0.015 g DAB in 10 ml of 0.1 M Tris buffer (pH 8.2) and illuminated by the Nikon 10 x Flour objective (0.50 NA) for 50 min. Photoconverted sections were washed in the phosphate buffer, and were processed for the transmission electron microscopy. The sections were then postfixed with 2% OsO4 in 0.1 M phosphate buffer. After that, the specimens were dehydrated through a graded series of ethanol and embedded in TAAB 812 resin. Semithin sections (about 1/Ira) were stained with 1% Toluidine blue, and thin sections (about
Fig. 2. An unstained semithin section of a solitary cell after photoconversion ofdil. Note that photoconversion produced a dark end product not only in a Merkel-like cell (arrow) but also in nerve fibers (arrowheads) passing in the lamina propia. Bar = 10/Jm.
T Nagai, H. Koyama/Neuroscience Letters 178 (1994) 1-4
3
Fig. 3. Ultrastructural features of the solitary cell shown in Fig. 2. Solid arrows and arrowheads point respectively to finger-like processes and a deep invagination of the nuclear envelope. Open arrows indicate patches of electron-dense plasma membrane. Bar = 1/lm.
cell had many dense-cored vesicles, finger-like processes and a large nucleus. Such ultrastructural features have been observed in the basal cell of the taste bud in axolotls [12], mudpuppies [1] and African clawed toads [13]. The basal cell in these amphibian taste buds has cytological characteristics similar to the Merkel cell, which is thought to be a tactile receptor in mammals [6]. Although the present ultrastructural analysis revealed no nerve endings on the solitary cell, the transneuronal labeling by diI suggests that the labeled solitary cells have close structural relationship with the IX nerve [4]. Thus, it may be assumable that the solitary cells are involved in mechanotransduction through the IX nerve of axolotls. We wish to thank The Indiana University Axolotl Colony for a continuous supply of axolotls. This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 06640887) to T.N. from the Ministry of Education, Science and Culture of Japan. [1] Delay, R.J. and Roper, S.D., Ultrastructure of taste cells and synapses in the mudpuppy Necturus maculosus, J. Comp. Neurol., 277 (1988) 268-280. [2] Diamond, J., Holmes, M. and Nurse, C.A., Are Merkel cellneurite reciprocal synapses involved in the initiation of tactile responses in salamander skin?, J. Physiol., 376 (1986) 101-120. [3] Farbman, A.I. and Yonkers, J.D., Fine structure of the taste bud
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
in the mud puppy, Necturus maculosus, Am. J. Anat., 131 (1971) 353-370. Godement, P., Vanselow, J., Thanos, S. and Bonhoeffer, F., A study in developing visual systems with a new method of staining neurons and their processes in fixed tissue, Development, 101 (1987) 697-713. Hanamori, T., Hirota, K. and Ishiko, N., Receptive fields and gustatory responsiveness of frog glossopharyngeal nerve, J. Gen. Physiol., 95 (1990) 1159-1182. lggo, A. and Muir, A.R., The structure and function of a slowly adapting touch corpuscle in hairy skin, J. Physiol., 200 (1969) 763 796. Maranto, A.R., Neuronal mapping: a photooxidation reaction makes Lucifer Yellow useful for electron microscopy, Science, 217 (1982) 953-955. Nagai, T. Transcellular labeling by diI demonstrates the glossopharyngeal innervation of taste buds in the lingual epithelium of the axolotl, J. Comp. Neurol., 31 (1993) 122-133. Samanen, D.W., and Bernard, R.A., Response properties of the glossopharyngeal taste system of the mud puppy (Necturus maculosus) I. General organization and whole nerve responses, J. Comp. Physiol., 143 (1981) 143 150. Sandell, J.H. and Masland, R.H., Photoconversion of some fluorescent markers to a diaminobenzidine product, J. Histochem. Cytochem., 36 (1988) 555-559. Takeuchi, H.-A., Masuda, T., and Nagai, T., Electrophysiological and behavioral studies of taste discrimination in the axolotl (Ambystoma mexieanum), Physiol. Behav., 56 (1994) 121-127. Toyoshima, K., Miyamoto, K. and Simamura, A., Fine structure of taste buds in the tongue, platal mucosa and gill arch of the axolotl, Ambystoma mexicanum, Okajimas Folia Anat. Jpn., 64 (1987) 99-110.
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7~ Nagai, H. Koyama/Neuroseience Letters 178 (1994) 1--4
[13] Toyoshima, K. and Shimamura, A., Comparative study of ultrastructures of the lateral-line organs and the palatal taste organs in the African clawed toad, Xenopus laevis, Anat. Rec., 204 (1982) 371 -381. [14] Toyoshima, K. and Shimamura, A., Monoamine-containing basal cells in the taste buds of the newt Trituruspyrrhogaster, Arch. Oral Biol., 32 (1987) 619-621.
[15] Yamane, S., Influence of ions and chemical substances on the response of the frog's tongue to mechanical stimulation, Comp. Biochem. Physiol. A, 61 (1978) 451459.
ELSEVIER
NEUROSCItNCE IETItRS
Ultras ructure of Merkel-like cells labeled with carbocyanine dye in the non-taste lingual epithelium of the axolotl T a k a t o s h i N a g a i a'*, H i r o m i c h i K o y a m a b aDepartment of Physiology, Teikyo University School of Medicine, Tokyo 173, Japan bDepartment of Anatomy, School of Medicine, Yokohama City University, Yokohama, Japan
Received 14 June 1994; Revised version received20 June 1994; Accepted 28 June 1994
Abstract
Fluorescent carbocyanine dye (diI), applied to the glossopharyngeal (IX) nerve of the axolotl, transneuronally labeled solitary cells in the non-taste lingual epithelium. With diaminobenzidine (DAB), the diI was photoconverted to a dark, electron-dense product. The labeled cell had a large nucleus with invaginations, dense-cored vesicles in the cytoplasm, and finger-like processes. These are reminiscent of morphological features of cutaneous Merkel cells, suggesting that solitary cells innervated by the IX nerve are associated with mechanosensory function of the IX nerve system. Key words." Mechanotransduction; Merkel cell; Salamander; Lingual epithelium; Fluorescent dyes; Photoconversion; Transmission electron microscopy
The glossopharyngeal (IX) nerve of the amphibian responds to chemical and mechanical stimuli applied to the lingual epithelium [5,9,11,15]. Chemical stimuli are transduced by taste receptor cells in the taste bud, whereas trangduction elements for mechanical stimuli remain unclear. One of the possible elements for mechanical transduction in the IX nerve responses is the basal cell in the taste bud, because the cell has cytological features similar to cutaneous Merkel cells [1,3,13,14] and is innervated by the IX nerve [8], although the function of the Merkel cell is controversial [2]. We recently examined the innervation pattern in the lingual epithelium of axolotls using transneuronal labeling with fluorescent carbocyanine dye, and found that the IX nerve innervates not only cells in the taste buds but also solitary cells in the non-taste lingual epithelium [8]. However, this study did not reveal cytological features of the solitary cells. Therefore, in the present study, fluorescence of solitary cells was photoconverted [7,10] into an electron-
*Corresponding author. Fax: (81) 3-5248-1415. 0304-3940194l$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved SSDI 0304-3940(94)00511-7
dense reaction product for subsequent electron microscopic observations. Juvenile axolotls, Ambystoma mexicanum, of the white strain were obtained from the Indiana University Axolotl Colony and raised in our laboratory. Three axolotls of about 7 month old (10 cm total length) were anesthetized in 0.2% MS222 (tricaine methanesulfonate; Sankyo) for 20-30 min and were perfused transcardially with a fixative containing 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). Glutaraldehyde level was kept low to avoid substantial background fluorescence. The tongues were dissected out, kept in the same fixative overnight, and transferred to the fixative without glutaraldehyde. A 2.5% solution ofdiI (Molecular Probes, Eugene, OR, U.S.A.) dissolved in 50% ethanol and 50% dimethyl sulfoxide was applied to the IX nerve stump. Methods of application of diI were described in detail elsewhere [8]. After the application of diI, the tongue was covered with 5% agar to prevent the accidental spread of the dye to the surrounding tissues and placed in the paraformaldehyde solution in a 37°C oven for 2 months. The tissue was then freed from the agar, reembedded in gelatin, hardened overnight in the same fixative and sectioned on a Microslicer
2
Z Nagai, H. Koyama/Neuroscience Letters 17~; (1994) 1 4
Fig. I. Photomicrographs of the dorsal surface of wholemounted lingual epithelium. A: fluorescent dil-labeled solitary cell (arrow) and taste buds (arrow heads) in the epithelium. The upper solitary cell in A is out of focus in B. B: photoconverted epithelium with dark end-product. Note corresponding solitary cell (arrow) and taste buds (arrowheads) in A and B. Bar = 100/Jm.
40 nm) were examined, usually without staining, at 75 kV in a Hitachi H-600 transmission electron microscope. If necessary, the thin sections were stained with uranyl acetate and lead citrate. DiI intensely labeled the nerve fibers beneath the lingual epithelium, as well as many taste buds and solitary cells which were distributed in the lingual epithelium (Fig. 1A). Labeled cells outside of the taste buds (nontaste epithelium) were easily located with fluorescent microscope, since labelings in the non-taste epithelium usually occurred in a solitary cell, not in a cell cluster like a taste bud [8]. In the presence of DAB, fluorescent illumination produced dark-brown reaction product (Fig. I B). Brightly fluorescent cells in the non-taste epithelium were turned into the darkest (arrow in Fig. 1). Thus, small tissues containing a photoconverted cell could easily be excised for subsequent light and electron microscopies. Labeled solitary cells were usually found in the deeper layer of the non-taste epithelium (Fig. 2). In the labeled solitary cells, the photoconversion product ofdiI occurred as patches of electron dense plasma membrane and outer nuclear envelope (Fig. 3). A large nucleus with an invagination was surrounded by a limb of cytoplasm which contained many vesicles. The cell had some finger-like processes extending into the neighboring cells. In the lingual epithelium of axolotls, taste bud cells form a rosette-like arrangement distinct from general epithelial cells, whereas solitary cells in the epithelium do not [8]. Therefore, without some kinds of labeling, it is difficult to examine a tissue containing solitary cells histologically. The dark brown end-product of diI photoconversion allowed us to locate the solitary cells. Electron microscopy revealed that the labeled solitary
(Dosaka EM Co., Ltd., Kyoto). Thick sections (200/tm) of the tissue were cut in the tangential plane to the dorsal surface of the tongue. Specimens were viewed by means of a Nikon fluorescence microscope equipped with an epi-illumination system (rhodamine filter set, G-2A). The sections were photoconverted with diaminobenzidine (DAB) to produce a dark, electron-dense product, following the procedure described by Sandell and Masland [10]. The sections were immersed in a solution of 0.015 g DAB in 10 ml of 0.1 M Tris buffer (pH 8.2) and illuminated by the Nikon 10 x Flour objective (0.50 NA) for 50 min. Photoconverted sections were washed in the phosphate buffer, and were processed for the transmission electron microscopy. The sections were then postfixed with 2% OsO4 in 0.1 M phosphate buffer. After that, the specimens were dehydrated through a graded series of ethanol and embedded in TAAB 812 resin. Semithin sections (about 1/Ira) were stained with 1% Toluidine blue, and thin sections (about
Fig. 2. An unstained semithin section of a solitary cell after photoconversion ofdil. Note that photoconversion produced a dark end product not only in a Merkel-like cell (arrow) but also in nerve fibers (arrowheads) passing in the lamina propia. Bar = 10/Jm.
T Nagai, H. Koyama/Neuroscience Letters 178 (1994) 1-4
3
Fig. 3. Ultrastructural features of the solitary cell shown in Fig. 2. Solid arrows and arrowheads point respectively to finger-like processes and a deep invagination of the nuclear envelope. Open arrows indicate patches of electron-dense plasma membrane. Bar = 1/lm.
cell had many dense-cored vesicles, finger-like processes and a large nucleus. Such ultrastructural features have been observed in the basal cell of the taste bud in axolotls [12], mudpuppies [1] and African clawed toads [13]. The basal cell in these amphibian taste buds has cytological characteristics similar to the Merkel cell, which is thought to be a tactile receptor in mammals [6]. Although the present ultrastructural analysis revealed no nerve endings on the solitary cell, the transneuronal labeling by diI suggests that the labeled solitary cells have close structural relationship with the IX nerve [4]. Thus, it may be assumable that the solitary cells are involved in mechanotransduction through the IX nerve of axolotls. We wish to thank The Indiana University Axolotl Colony for a continuous supply of axolotls. This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 06640887) to T.N. from the Ministry of Education, Science and Culture of Japan. [1] Delay, R.J. and Roper, S.D., Ultrastructure of taste cells and synapses in the mudpuppy Necturus maculosus, J. Comp. Neurol., 277 (1988) 268-280. [2] Diamond, J., Holmes, M. and Nurse, C.A., Are Merkel cellneurite reciprocal synapses involved in the initiation of tactile responses in salamander skin?, J. Physiol., 376 (1986) 101-120. [3] Farbman, A.I. and Yonkers, J.D., Fine structure of the taste bud
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
in the mud puppy, Necturus maculosus, Am. J. Anat., 131 (1971) 353-370. Godement, P., Vanselow, J., Thanos, S. and Bonhoeffer, F., A study in developing visual systems with a new method of staining neurons and their processes in fixed tissue, Development, 101 (1987) 697-713. Hanamori, T., Hirota, K. and Ishiko, N., Receptive fields and gustatory responsiveness of frog glossopharyngeal nerve, J. Gen. Physiol., 95 (1990) 1159-1182. lggo, A. and Muir, A.R., The structure and function of a slowly adapting touch corpuscle in hairy skin, J. Physiol., 200 (1969) 763 796. Maranto, A.R., Neuronal mapping: a photooxidation reaction makes Lucifer Yellow useful for electron microscopy, Science, 217 (1982) 953-955. Nagai, T. Transcellular labeling by diI demonstrates the glossopharyngeal innervation of taste buds in the lingual epithelium of the axolotl, J. Comp. Neurol., 31 (1993) 122-133. Samanen, D.W., and Bernard, R.A., Response properties of the glossopharyngeal taste system of the mud puppy (Necturus maculosus) I. General organization and whole nerve responses, J. Comp. Physiol., 143 (1981) 143 150. Sandell, J.H. and Masland, R.H., Photoconversion of some fluorescent markers to a diaminobenzidine product, J. Histochem. Cytochem., 36 (1988) 555-559. Takeuchi, H.-A., Masuda, T., and Nagai, T., Electrophysiological and behavioral studies of taste discrimination in the axolotl (Ambystoma mexieanum), Physiol. Behav., 56 (1994) 121-127. Toyoshima, K., Miyamoto, K. and Simamura, A., Fine structure of taste buds in the tongue, platal mucosa and gill arch of the axolotl, Ambystoma mexicanum, Okajimas Folia Anat. Jpn., 64 (1987) 99-110.
4
7~ Nagai, H. Koyama/Neuroseience Letters 178 (1994) 1--4
[13] Toyoshima, K. and Shimamura, A., Comparative study of ultrastructures of the lateral-line organs and the palatal taste organs in the African clawed toad, Xenopus laevis, Anat. Rec., 204 (1982) 371 -381. [14] Toyoshima, K. and Shimamura, A., Monoamine-containing basal cells in the taste buds of the newt Trituruspyrrhogaster, Arch. Oral Biol., 32 (1987) 619-621.
[15] Yamane, S., Influence of ions and chemical substances on the response of the frog's tongue to mechanical stimulation, Comp. Biochem. Physiol. A, 61 (1978) 451459.