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Ontogeny of regulatory neuropeptides in the bullfrog taste organ
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 96 (1996) 285-289
Short communication
Ontogeny of regulatory neuropeptides in the bullfrog taste organ Tatsumi Kusakabe a,*, Hideki Matsuda b Tadashi Kawakami c Noriko Syoui d Katsuyoshi Kurihara d, Toshifumi Takenaka e, Hajime Sawada a a Department of Anatomy, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan b Department of Otorhinolaryngology, Yokohama City University School of Medicine, Yokohama 236, Japan c Department of Physiology, Kitasato University School of Medicine, Sagamihara 228, Japan d Department of Legal Medicine, Kitasato University School of Medicine, Sagamihara 228, Japan e Department of Physiology, Yokohama City University School of Medicine, Yokohama 236, Japan Accepted 11 June 1996
Abstract
During metamorphic stages (stages XX--XXV), the first appearance of nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), galanin (GAL), and neuropeptide Y (NP¥) in the bullfrog taste organs was different for each substance. CGRP fibers appeared first in association with the immature taste organs at stage XX. Up to stage XXV, the taste organs, epithelial disks, are close to their adult form, and SP, VIP, and GAL fibers appeared within them. Throughout these stages, NPY fibers were absent, and no taste cells had immunoreactivity of the five neuropeptides. The present findings and previous physiological studies suggest that the immature taste organs in metamorphic stages already function as chemical and mechanical receptors and that these sensory mechanisms are under the control of peptidergic innervation. Keywords: Taste organ; Neuropel:,tide; Ontogeny; Immunohistochemistry; Bullfrog
In adult anurans, the dorsal lingual surface has a velvety appearance caused by two types of lingual papillae, filiform and fungiform papillae [5]. On their free tops, the taste organs are recognized as epithelial disks [1,2]. The epithelial disks, which correspond to the mammalian taste buds, are composed of two types of cells, sensory and associate cells, and are surrounded by ciliated epithelial cells. The epithelial disks are richly innervated [11], and the sensory nerve endings establish synaptic contact with the sensory cells [2,12,14]. Nerve fibers containing :aeuropeptide such as substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP) have been immunohistochemically demonstrated in the adult bullfrog fungiform papillae [3,7]. Recently, Kusakabe et al. [9] demonstrated that all SP fibers in the connective tissue core of the fungiform and filiform papillae, within the epithelial disks and the epithelial ciliated cells on the top of the fungiform papillae, and glands and blood vessels in the lamina propria of the tongue show coexistence with CGRP,
* Corresponding author. Fax: (81) (45) 787-2509.
and demonstrated that SP and VIP fibers and SP and galanin (GAL) fibers are two intertwined fibers within the same thin nerve bundle. On this basis, Kusakabe et al. [9] suggested that the chemoreceptor function of the bullfrog taste organs may be under the control of complicated peptidergic innervation. To clarify the role of neuropeptides in the amphibian gustatory mechanism, it is important to examine the occurrence and distribution of the peptidergic innervation during development, because after metamorphosis the juveniles live both in water and on land, although the larvae live only in water. Therefore, in the present study, the ontogenesis of SP, CGRP, VIP, NPY, and GAL, which are localized in the adult bullfrog taste organs [9], was examined during metamorphosis. Twenty-five larvae and five adult bullfrogs, Rana catesbeiana, were used in this study. The larvae were kept in glass tanks at 23-25°C. Stages during larval development were referred to those in the normal development (stages I - X X V ) of Rana pipiens larvae described by Taylor and Kollros [13]. Larvae were killed at the following stages: XIX, XX, XXI, XXII, XXIII, XXIV, and XXV. The day just before the cloacal tall piece was completely resorbed was taken as stage XXV. At least three larvae of
0165-3806/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S 0 1 6 5 - 3 8 0 6 ( 9 6 ) 0 0 l 21-6
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T. Kusakabe et a l . / Developmental Brain Research 96 (1996) 285-289
each of the seven stages were examined. After anesthesia by immersion in a 1% aqueous solution of tricainemethanesulfate (MS-222) for a few minutes, the thoracic cavity was opened to expose the heart. Through a thin nylon tube inserted into the aortic trunk, the animal was briefly perfused with heparinized (1 I U / m l ) 0.1 M phosphate buffered saline (PBS), pH 7.4, and then with freshly prepared Zamboni's fixative solution (4% paraformaldehyde, 0.2% picric acid in 0.1 M PBS, pH 7.4) for 10 min. The tongues were transversely cut into thin slices, and immersed in the same fixative for an additional 8 h at 4°C. After a brief wash with PBS, the specimens were transferred to 30% sucrose in PBS, and kept there overnight at 4°C. The specimens were then sectioned serially at 15 /xm on a cryostat, and mounted on poly-L-lysine coated slides. Some sections of each stage were stained with hematoxylin-eosin for general histology. The sections were immunohistochemically stained for SP, CGRP, GAL (Cambridge), VIP, NPY (Incstar) with the peroxidase-antiperoxidase (PAP) method. The process of immunostaining was detailed in a previous report [8]. Primary antisera were preincubated with 50 /xM of the respective peptide. The absorbed antiserum was used for incubation of the section followed by incubation with the secondary antiserum to test the specificity of the primary antiserum. At the late premetamorphic stage, stage XIX, the dorsal surface of the tongue was covered with the epithelium composed of inner columnar and outer cuboidal cells (Fig. 1A), on which two finger-like premetamorphic processes still remained. Up to this stage, no specific immunoreactivity was detected within the epithelium (Fig. 2A), although
a few CGRP immunoreactive fibers were already scattered in the connective tissue of the lamina propria (Fig. 2B). The cloacal tail pieces of the larvae begin to regress on stage XX, and are entirely resorbed on stage XXV to complete metamorphosis. Through these metamorphic stages, the morphogenesis of the epithelial disk made progress as previously reported by scanning electron microscopy [10]. At stage XX, the thickness of the epithelium increased, and the lamina propria began to come into the epithelium (Fig. 1B). At the surface layer of the epithelium, several epithelial cells gathered to form oval cell clusters (Fig. 1B). At this stage, CGRP (Fig. 2C), SP, and VIP immunoreactive fibers were first found to enter the epithelium, although the number of these fibers was small. The CGRP fibers reached the bottom of the cell clusters (Fig. 2C), and some branches were recognized within the cell clusters. At stage XXI, the lamina propria penetrated into the epithelium to form the cylindrical connective tissue core of the primitive fungiform papillae, but the cell cluster remained unchanged (Fig. 1C). At this stage, a few GAL fibers first occurred in the connective tissue core of fungiform papillae, and some of them reached the bottom of the cell clusters (Fig. 2D). Between stages XXII and XXIV, the primitive epithelial disks, which are composed of an outer layer of columnar supporting cells, and a few inner layers of cuboidal taste cells, were distinguished in the center of the top of the fungiform papillae (Fig. 1D,E). The ciliated cells were confined to the edge of the apical surface of the fungiform papillae. Many glands, which consist of the invagination of the epithelium, were located in the lamina propria (Fig. 1D,E). During these stages, the
XXl
Fig. 1. Light micrographsshowing the morphogenesisof the bullfrogtaste organ at stage (St.) XIX (A), XX (B), XXI (C), XXII (D), XXIV(E), and XXV (F). Three arrows in B indicate the cell clusters, and the arrowheadsin D-F indicate the epithelial discs, e, epithelium;fi, filiforrnpapillae; fu, fungiform papillae; g, glands; lp, lamina propria. Bar = 100 /xrn.
T. Kusakabe et al. / Developmental Brain Research 96 (1996) 285-289
number of CGRP fibers which penetrated into the epithelial disks and ciliated cells increased (Fig. 2E,F), but there was no conspicuous change in the distribution and abundance of SP and VIP fibers. NPY fibers were still absent. At stage XXIV, SP and GAL fibers were first detected within the epithelial disks and ciliated cells (Fig. 2G). At stage XXV (Fig. IF), the final stage of metamorphosis, the fungiform and filiform papillae were close to the adult form previously described by Kusakabe et al. [9]. In transverse sections through the middle of the fungiform papillae, the epithelial disks were also close to the cell arrangement in the adults as previously demonstrated [9]. At this stage, VIP fibers in the connective tissue of both
287
papillae and around the glands increased in number (Fig. 2H). NPY fibers were still absent. After metamorphosis, SP (Fig. 2I), CGRP, VIP (Fig. 2J) and GAL fibers (Fig. 2K) within the epithelial disk quickly increased in number. Through all stages, no taste cells had immunoreactivity of the five neuropeptides in the epithelial disk. Table 1 summarizes the distribution and relative abundance of the five neuropeptides during metamorphosis and in the adults of the bullfrog lingual epithelium. No immunoreactivity for the five neuropeptides was detected in the sections incubated with preabsorbed antisera. During morphogenesis of the bullfrog taste organ, the time of appearance of the five neuropeptides was different
Table 1 Distribution a n d r e l a t i v e a b u n d a n c e o f i m m u n o r e a c t i v e n e r v e fibers in the b u l l f r o g lingual e p i t h e l i u m S t a g e [13] XIX SP
F u n g i f o r m p. ED CiC LP F i l i f o r m p. E LP Glands
CGRP
VIP
E LP Glands F u n g i f o r m p.
NPY
GAL
ED CiC LP F i l i f o r m p. E LP Glands
XXII
XXIII
CCI( - )
(- )
+
E ( _+ )
LP( - )
LP( + )
(+ )
CCI(+)
(+)
E( + )
LP( + )
)
-
E( + )
. _
+ +
+ + + +
.
.
.
+
. + +
+
+ +
+ +
+ +
+ +
+
+ ++
+ ++
+ ++
+ + + +++
+ +
+ + + +
-
-
___
+ + +
+
+
_+ ++
+ ++++
++ + + +
+++ + + + +
(-)
.
.
.
.
+ +
_ +
. + + . . .
.
.
.
+ + .
.
. .
. + +
. .
.
.
. .
+ +
(- )
LP( - )
(- ) )
(- )
E( - )
LP(- )
+ + + +++
(+ )
E( - )
LP(- )
+ +
+ ++
+
CCI(-)
E( - )
+ +
+ +
-
(+ )
-
+
+
(- )
LP(+)
CCI(
-
(+ )
E( - )
LP( - )
Adult
. +
E( - )
LP( - )
XXV
(+ )
LP( + ) CCI(
XXIV
( _+ )
E(- )
ED CiC LP F i l i f o r m p. E LP Glands F u n g i f o r m p. ED CiC LP F i l i f o r m p. l LP Glands F u n g i f o r m p.
XXI
E(-)
F u n g i f o r m p. ED CiC LP F i l i f o r m p.
XX
. .
.
.
.
.
+
. -
.
.
++
_+
+
+ + ++
+ + +++
++ + +
+++ + + +
. + + ++
(- )
(+)
. + +
.
. ++ + +
.
. ++ + +
G r a d i n g o f f r e q u e n c y o f i m m u n o r e a c t i v e n e r v e fibers: - absent, + v e r y f e w , + f e w , + + m o d e r a t e n u m b e r , + + + m a n y , + + + + abundant. B e c a u s e the epithelial disk is n o t w e l l e s t a b l i s h e d until stage X X I I , the classification o f lingual m u c o s a is different in stages X I X , X X , a n d X X I . CC1, cell cluster; CiC, ciliated cell; E, e p i t h e l i u m ; E D , epithelial disk; LP, l a m i n a propria.
288
T. Kusakabe et al. / Deuelopmental Brain Research 96 (1996) 285-289
for each. First, CGRP immunoreactive fibers appeared in association with the cell clusters in the tongue rudiments at stage XX. At this stage, however, the morphogenesis of epithelial disks was not yet been completed. Nevertheless, in an electrophysiological study [4] the afferent discharges generated in the glossopharyngeal nerve were first recorded by both chemical and tactile stimuli of the tongue rudiments at stage XX. This clearly suggests that the immature taste organs, cell clusters, at this stage have already begun to function as taste and tactile receptors, and that CGRP containing fibers may be involved in controlling these sensory mechanisms. This suggestion is also supported by the fact that the taste organs of the caudates, which are classified as phylogenetically lower than the frogs, are represented as many barrel-shaped cell clusters within the dorsal tongue epithelium. Thereafter, SP, VIP, and G A L fibers appeared in association with the epithelial disks at stages XXIV or XXV,
when the epithelial disks are close to their adult form. According to the electrophysiological study described above [4], the afferent discharges of the glossopharyngeal nerve produced by chemical and mechanical stimuli at these stages increase, and these responses begin to resemble those of the adult. In addition, the glossopharyngealhypoglossal reflex [6], a fundamental reflex for the intake of food, has been confirmed at these stages [4]. Although the morphogenesis of the epithelial disks has not yet been completed at these stages, it appears that the immature epithelial disks already function as taste and tactile organs, and that CGRP, SP, VIP, and G A L containing fibers may take part in the chemical and mechanical reception of the immature disks. On the other hand, NPY fibers probably do not participate in taste sensory mechanisms during metamorphic stages, because NPY fibers were not detected in association with taste organs. In conclusion, the present immunohistochemical results
Fig. 2. CGRP, GAL, SP, and VIP immunoreactive fibers in the bullfrog developing taste organs. Stages (St.) during development are indicated by roman numerals. A: no SP immunoreactivity within the epithelium (e) at St. XIX. B: a few CGRP fibers in the lamina propria (lp) at St. XIX. C: CGRP fibers associated with the cell cluster (arrowhead) at St. XX. D: first appearance of GAL fibers in the fungiform papillae at St. XXI. E and F: CGRP fibers (arrows) associated with the epithelial discs (arrowhead) at St. XXII (E) and St. XXIV (F). G: SP fibers (arrows) within the epithelial disk (arrowhead) at St. XXIV. H: VIP fibers in the fungiform (fu) and filiform (fi) papillae and around glands (g) at St. XXV. I, J, and K: SP (I), VIP (J), and GAL (K) fibers in the fungiform papillae (fu) of adults (Ad). Bars = 100 /~m.
T. Kusakabe et al. / Developmental Brain Research 96 (1996) 285-289
and p r e v i o u s p h y s i o l o g i c a l studies suggest that the i m m a ture taste organs in m e t a m o r p h i c stages already function as taste and tactile receptors and that these sensory m e c h a nisms are under the contJ:ol o f c o m p l i c a t e d peptidergic innervation.
Acknowledgements W e are grateful to Dr. R.C. Goris o f the D e p a r t m e n t o f A n a t o m y , Y o k o h a m a City University School o f M e d i c i n e , for his help in editing the manuscript. T h e present w o r k was supported by grants in support o f the p r o m o t i o n o f research at Y o k o h a m a City University, and Grant-in-aid 0 5 6 7 0 0 2 4 f r o m the M i n i s t r y o f Education, S c i e n c e and Culture, Japan.
References [1] Delian, R.S. and Graziadei, P.P.C., Functional anatomy of frog's taste organs, Experientia, 27 (1971) 823-826. [2] Graziadei, P.P.C. and Delian, R.S., The ultrastructure of frog's taste organs, Acta Anat., 80 (1971) 563-603. [3] Hirata, K. and Kanaseki, T., Substance P-like immunoreactive fibers in the frog taste organs, Experientia, 43 (1987) 386-389. [4] Honda, E., Hirakawa, T., Nakamura, S. and Nakahara, S., Tactile
289
and taste sensory maturation of the frog larvae tongue, Brain Res., 595 (1992) 74-78. [5] Jaeger, C.B. and Hillman, D.E., Morphology of gustatory organs. In: R. Llians and W. Precht (Eds.), Frog Neurobiology, Springer-Verlag, Heidelberg, 1976, pp. 587-606. [6] Kozai, H., Reflex discharge of hypoglossal nerve of frogs, J. Kyushu Dent. Soc., 29 (1976) 210-221. [7] Kuramoto, H., An immunohistochemical study of cellular and nervous elements in the taste organ of the bullfrog, Rana catesbeiana, Arch. Histol. CytoL, 51 (1988) 205-221. [8] Kusakabe, T., Anglade, P. and Tsuji, S., Localization of substance P, CGRP, VIP, neuropeptide Y, and somatostatin immunoreactive nerve fibers in the carotid labyrinths of some amphibian species, Histochemistry, 96 (1991) 255-260. [9] Kusakabe, T., Kawakami, T., Ono, M., Sawada, H. and Takenaka, T., Distribution of peptidergic nerve fibers in bullfrog lingual papillae demonstrated by a combination of double immunofluorescence labelling and a multiple dye filter, Histochem. J., 28 (1996) 289-297. [10] Nomura, S., Studies on the development and differentiation of frog's taste organs with scanning and transmission electron microscopes, J. Hiroshima Univ. Dent. Soc., 8 (1976) 182-212. [11] Rapuzzi, G. and Casella, C., Innervation of the fungiform papillae in the frog tongue, J. NeurophysioL, 28 (1965) 154-165. [12] Stensaas, L.J., The fine structure of the fungiform papillae and epithelium of the tongue of South American toad, Calyptocephalella gayi, Am. J. Anat., 131 (1971) 443-462. [13] Taylor, A.C. and Kollros, J.J., Stages in the normal development of Rana pipiens larvae, Anat. Rec., 94 (1946) 7-25. [14] Uga, S. and Hama, K., Electron microscopic study on the synaptic region of the taste organ of carps and frogs, J. Electronmicrosc., 16 (1967) 269-276.
ELSEVIER
Developmental Brain Research 96 (1996) 285-289
Short communication
Ontogeny of regulatory neuropeptides in the bullfrog taste organ Tatsumi Kusakabe a,*, Hideki Matsuda b Tadashi Kawakami c Noriko Syoui d Katsuyoshi Kurihara d, Toshifumi Takenaka e, Hajime Sawada a a Department of Anatomy, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan b Department of Otorhinolaryngology, Yokohama City University School of Medicine, Yokohama 236, Japan c Department of Physiology, Kitasato University School of Medicine, Sagamihara 228, Japan d Department of Legal Medicine, Kitasato University School of Medicine, Sagamihara 228, Japan e Department of Physiology, Yokohama City University School of Medicine, Yokohama 236, Japan Accepted 11 June 1996
Abstract
During metamorphic stages (stages XX--XXV), the first appearance of nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), galanin (GAL), and neuropeptide Y (NP¥) in the bullfrog taste organs was different for each substance. CGRP fibers appeared first in association with the immature taste organs at stage XX. Up to stage XXV, the taste organs, epithelial disks, are close to their adult form, and SP, VIP, and GAL fibers appeared within them. Throughout these stages, NPY fibers were absent, and no taste cells had immunoreactivity of the five neuropeptides. The present findings and previous physiological studies suggest that the immature taste organs in metamorphic stages already function as chemical and mechanical receptors and that these sensory mechanisms are under the control of peptidergic innervation. Keywords: Taste organ; Neuropel:,tide; Ontogeny; Immunohistochemistry; Bullfrog
In adult anurans, the dorsal lingual surface has a velvety appearance caused by two types of lingual papillae, filiform and fungiform papillae [5]. On their free tops, the taste organs are recognized as epithelial disks [1,2]. The epithelial disks, which correspond to the mammalian taste buds, are composed of two types of cells, sensory and associate cells, and are surrounded by ciliated epithelial cells. The epithelial disks are richly innervated [11], and the sensory nerve endings establish synaptic contact with the sensory cells [2,12,14]. Nerve fibers containing :aeuropeptide such as substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP) have been immunohistochemically demonstrated in the adult bullfrog fungiform papillae [3,7]. Recently, Kusakabe et al. [9] demonstrated that all SP fibers in the connective tissue core of the fungiform and filiform papillae, within the epithelial disks and the epithelial ciliated cells on the top of the fungiform papillae, and glands and blood vessels in the lamina propria of the tongue show coexistence with CGRP,
* Corresponding author. Fax: (81) (45) 787-2509.
and demonstrated that SP and VIP fibers and SP and galanin (GAL) fibers are two intertwined fibers within the same thin nerve bundle. On this basis, Kusakabe et al. [9] suggested that the chemoreceptor function of the bullfrog taste organs may be under the control of complicated peptidergic innervation. To clarify the role of neuropeptides in the amphibian gustatory mechanism, it is important to examine the occurrence and distribution of the peptidergic innervation during development, because after metamorphosis the juveniles live both in water and on land, although the larvae live only in water. Therefore, in the present study, the ontogenesis of SP, CGRP, VIP, NPY, and GAL, which are localized in the adult bullfrog taste organs [9], was examined during metamorphosis. Twenty-five larvae and five adult bullfrogs, Rana catesbeiana, were used in this study. The larvae were kept in glass tanks at 23-25°C. Stages during larval development were referred to those in the normal development (stages I - X X V ) of Rana pipiens larvae described by Taylor and Kollros [13]. Larvae were killed at the following stages: XIX, XX, XXI, XXII, XXIII, XXIV, and XXV. The day just before the cloacal tall piece was completely resorbed was taken as stage XXV. At least three larvae of
0165-3806/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S 0 1 6 5 - 3 8 0 6 ( 9 6 ) 0 0 l 21-6
286
T. Kusakabe et a l . / Developmental Brain Research 96 (1996) 285-289
each of the seven stages were examined. After anesthesia by immersion in a 1% aqueous solution of tricainemethanesulfate (MS-222) for a few minutes, the thoracic cavity was opened to expose the heart. Through a thin nylon tube inserted into the aortic trunk, the animal was briefly perfused with heparinized (1 I U / m l ) 0.1 M phosphate buffered saline (PBS), pH 7.4, and then with freshly prepared Zamboni's fixative solution (4% paraformaldehyde, 0.2% picric acid in 0.1 M PBS, pH 7.4) for 10 min. The tongues were transversely cut into thin slices, and immersed in the same fixative for an additional 8 h at 4°C. After a brief wash with PBS, the specimens were transferred to 30% sucrose in PBS, and kept there overnight at 4°C. The specimens were then sectioned serially at 15 /xm on a cryostat, and mounted on poly-L-lysine coated slides. Some sections of each stage were stained with hematoxylin-eosin for general histology. The sections were immunohistochemically stained for SP, CGRP, GAL (Cambridge), VIP, NPY (Incstar) with the peroxidase-antiperoxidase (PAP) method. The process of immunostaining was detailed in a previous report [8]. Primary antisera were preincubated with 50 /xM of the respective peptide. The absorbed antiserum was used for incubation of the section followed by incubation with the secondary antiserum to test the specificity of the primary antiserum. At the late premetamorphic stage, stage XIX, the dorsal surface of the tongue was covered with the epithelium composed of inner columnar and outer cuboidal cells (Fig. 1A), on which two finger-like premetamorphic processes still remained. Up to this stage, no specific immunoreactivity was detected within the epithelium (Fig. 2A), although
a few CGRP immunoreactive fibers were already scattered in the connective tissue of the lamina propria (Fig. 2B). The cloacal tail pieces of the larvae begin to regress on stage XX, and are entirely resorbed on stage XXV to complete metamorphosis. Through these metamorphic stages, the morphogenesis of the epithelial disk made progress as previously reported by scanning electron microscopy [10]. At stage XX, the thickness of the epithelium increased, and the lamina propria began to come into the epithelium (Fig. 1B). At the surface layer of the epithelium, several epithelial cells gathered to form oval cell clusters (Fig. 1B). At this stage, CGRP (Fig. 2C), SP, and VIP immunoreactive fibers were first found to enter the epithelium, although the number of these fibers was small. The CGRP fibers reached the bottom of the cell clusters (Fig. 2C), and some branches were recognized within the cell clusters. At stage XXI, the lamina propria penetrated into the epithelium to form the cylindrical connective tissue core of the primitive fungiform papillae, but the cell cluster remained unchanged (Fig. 1C). At this stage, a few GAL fibers first occurred in the connective tissue core of fungiform papillae, and some of them reached the bottom of the cell clusters (Fig. 2D). Between stages XXII and XXIV, the primitive epithelial disks, which are composed of an outer layer of columnar supporting cells, and a few inner layers of cuboidal taste cells, were distinguished in the center of the top of the fungiform papillae (Fig. 1D,E). The ciliated cells were confined to the edge of the apical surface of the fungiform papillae. Many glands, which consist of the invagination of the epithelium, were located in the lamina propria (Fig. 1D,E). During these stages, the
XXl
Fig. 1. Light micrographsshowing the morphogenesisof the bullfrogtaste organ at stage (St.) XIX (A), XX (B), XXI (C), XXII (D), XXIV(E), and XXV (F). Three arrows in B indicate the cell clusters, and the arrowheadsin D-F indicate the epithelial discs, e, epithelium;fi, filiforrnpapillae; fu, fungiform papillae; g, glands; lp, lamina propria. Bar = 100 /xrn.
T. Kusakabe et al. / Developmental Brain Research 96 (1996) 285-289
number of CGRP fibers which penetrated into the epithelial disks and ciliated cells increased (Fig. 2E,F), but there was no conspicuous change in the distribution and abundance of SP and VIP fibers. NPY fibers were still absent. At stage XXIV, SP and GAL fibers were first detected within the epithelial disks and ciliated cells (Fig. 2G). At stage XXV (Fig. IF), the final stage of metamorphosis, the fungiform and filiform papillae were close to the adult form previously described by Kusakabe et al. [9]. In transverse sections through the middle of the fungiform papillae, the epithelial disks were also close to the cell arrangement in the adults as previously demonstrated [9]. At this stage, VIP fibers in the connective tissue of both
287
papillae and around the glands increased in number (Fig. 2H). NPY fibers were still absent. After metamorphosis, SP (Fig. 2I), CGRP, VIP (Fig. 2J) and GAL fibers (Fig. 2K) within the epithelial disk quickly increased in number. Through all stages, no taste cells had immunoreactivity of the five neuropeptides in the epithelial disk. Table 1 summarizes the distribution and relative abundance of the five neuropeptides during metamorphosis and in the adults of the bullfrog lingual epithelium. No immunoreactivity for the five neuropeptides was detected in the sections incubated with preabsorbed antisera. During morphogenesis of the bullfrog taste organ, the time of appearance of the five neuropeptides was different
Table 1 Distribution a n d r e l a t i v e a b u n d a n c e o f i m m u n o r e a c t i v e n e r v e fibers in the b u l l f r o g lingual e p i t h e l i u m S t a g e [13] XIX SP
F u n g i f o r m p. ED CiC LP F i l i f o r m p. E LP Glands
CGRP
VIP
E LP Glands F u n g i f o r m p.
NPY
GAL
ED CiC LP F i l i f o r m p. E LP Glands
XXII
XXIII
CCI( - )
(- )
+
E ( _+ )
LP( - )
LP( + )
(+ )
CCI(+)
(+)
E( + )
LP( + )
)
-
E( + )
. _
+ +
+ + + +
.
.
.
+
. + +
+
+ +
+ +
+ +
+ +
+
+ ++
+ ++
+ ++
+ + + +++
+ +
+ + + +
-
-
___
+ + +
+
+
_+ ++
+ ++++
++ + + +
+++ + + + +
(-)
.
.
.
.
+ +
_ +
. + + . . .
.
.
.
+ + .
.
. .
. + +
. .
.
.
. .
+ +
(- )
LP( - )
(- ) )
(- )
E( - )
LP(- )
+ + + +++
(+ )
E( - )
LP(- )
+ +
+ ++
+
CCI(-)
E( - )
+ +
+ +
-
(+ )
-
+
+
(- )
LP(+)
CCI(
-
(+ )
E( - )
LP( - )
Adult
. +
E( - )
LP( - )
XXV
(+ )
LP( + ) CCI(
XXIV
( _+ )
E(- )
ED CiC LP F i l i f o r m p. E LP Glands F u n g i f o r m p. ED CiC LP F i l i f o r m p. l LP Glands F u n g i f o r m p.
XXI
E(-)
F u n g i f o r m p. ED CiC LP F i l i f o r m p.
XX
. .
.
.
.
.
+
. -
.
.
++
_+
+
+ + ++
+ + +++
++ + +
+++ + + +
. + + ++
(- )
(+)
. + +
.
. ++ + +
.
. ++ + +
G r a d i n g o f f r e q u e n c y o f i m m u n o r e a c t i v e n e r v e fibers: - absent, + v e r y f e w , + f e w , + + m o d e r a t e n u m b e r , + + + m a n y , + + + + abundant. B e c a u s e the epithelial disk is n o t w e l l e s t a b l i s h e d until stage X X I I , the classification o f lingual m u c o s a is different in stages X I X , X X , a n d X X I . CC1, cell cluster; CiC, ciliated cell; E, e p i t h e l i u m ; E D , epithelial disk; LP, l a m i n a propria.
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T. Kusakabe et al. / Deuelopmental Brain Research 96 (1996) 285-289
for each. First, CGRP immunoreactive fibers appeared in association with the cell clusters in the tongue rudiments at stage XX. At this stage, however, the morphogenesis of epithelial disks was not yet been completed. Nevertheless, in an electrophysiological study [4] the afferent discharges generated in the glossopharyngeal nerve were first recorded by both chemical and tactile stimuli of the tongue rudiments at stage XX. This clearly suggests that the immature taste organs, cell clusters, at this stage have already begun to function as taste and tactile receptors, and that CGRP containing fibers may be involved in controlling these sensory mechanisms. This suggestion is also supported by the fact that the taste organs of the caudates, which are classified as phylogenetically lower than the frogs, are represented as many barrel-shaped cell clusters within the dorsal tongue epithelium. Thereafter, SP, VIP, and G A L fibers appeared in association with the epithelial disks at stages XXIV or XXV,
when the epithelial disks are close to their adult form. According to the electrophysiological study described above [4], the afferent discharges of the glossopharyngeal nerve produced by chemical and mechanical stimuli at these stages increase, and these responses begin to resemble those of the adult. In addition, the glossopharyngealhypoglossal reflex [6], a fundamental reflex for the intake of food, has been confirmed at these stages [4]. Although the morphogenesis of the epithelial disks has not yet been completed at these stages, it appears that the immature epithelial disks already function as taste and tactile organs, and that CGRP, SP, VIP, and G A L containing fibers may take part in the chemical and mechanical reception of the immature disks. On the other hand, NPY fibers probably do not participate in taste sensory mechanisms during metamorphic stages, because NPY fibers were not detected in association with taste organs. In conclusion, the present immunohistochemical results
Fig. 2. CGRP, GAL, SP, and VIP immunoreactive fibers in the bullfrog developing taste organs. Stages (St.) during development are indicated by roman numerals. A: no SP immunoreactivity within the epithelium (e) at St. XIX. B: a few CGRP fibers in the lamina propria (lp) at St. XIX. C: CGRP fibers associated with the cell cluster (arrowhead) at St. XX. D: first appearance of GAL fibers in the fungiform papillae at St. XXI. E and F: CGRP fibers (arrows) associated with the epithelial discs (arrowhead) at St. XXII (E) and St. XXIV (F). G: SP fibers (arrows) within the epithelial disk (arrowhead) at St. XXIV. H: VIP fibers in the fungiform (fu) and filiform (fi) papillae and around glands (g) at St. XXV. I, J, and K: SP (I), VIP (J), and GAL (K) fibers in the fungiform papillae (fu) of adults (Ad). Bars = 100 /~m.
T. Kusakabe et al. / Developmental Brain Research 96 (1996) 285-289
and p r e v i o u s p h y s i o l o g i c a l studies suggest that the i m m a ture taste organs in m e t a m o r p h i c stages already function as taste and tactile receptors and that these sensory m e c h a nisms are under the contJ:ol o f c o m p l i c a t e d peptidergic innervation.
Acknowledgements W e are grateful to Dr. R.C. Goris o f the D e p a r t m e n t o f A n a t o m y , Y o k o h a m a City University School o f M e d i c i n e , for his help in editing the manuscript. T h e present w o r k was supported by grants in support o f the p r o m o t i o n o f research at Y o k o h a m a City University, and Grant-in-aid 0 5 6 7 0 0 2 4 f r o m the M i n i s t r y o f Education, S c i e n c e and Culture, Japan.
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and taste sensory maturation of the frog larvae tongue, Brain Res., 595 (1992) 74-78. [5] Jaeger, C.B. and Hillman, D.E., Morphology of gustatory organs. In: R. Llians and W. Precht (Eds.), Frog Neurobiology, Springer-Verlag, Heidelberg, 1976, pp. 587-606. [6] Kozai, H., Reflex discharge of hypoglossal nerve of frogs, J. Kyushu Dent. Soc., 29 (1976) 210-221. [7] Kuramoto, H., An immunohistochemical study of cellular and nervous elements in the taste organ of the bullfrog, Rana catesbeiana, Arch. Histol. CytoL, 51 (1988) 205-221. [8] Kusakabe, T., Anglade, P. and Tsuji, S., Localization of substance P, CGRP, VIP, neuropeptide Y, and somatostatin immunoreactive nerve fibers in the carotid labyrinths of some amphibian species, Histochemistry, 96 (1991) 255-260. [9] Kusakabe, T., Kawakami, T., Ono, M., Sawada, H. and Takenaka, T., Distribution of peptidergic nerve fibers in bullfrog lingual papillae demonstrated by a combination of double immunofluorescence labelling and a multiple dye filter, Histochem. J., 28 (1996) 289-297. [10] Nomura, S., Studies on the development and differentiation of frog's taste organs with scanning and transmission electron microscopes, J. Hiroshima Univ. Dent. Soc., 8 (1976) 182-212. [11] Rapuzzi, G. and Casella, C., Innervation of the fungiform papillae in the frog tongue, J. NeurophysioL, 28 (1965) 154-165. [12] Stensaas, L.J., The fine structure of the fungiform papillae and epithelium of the tongue of South American toad, Calyptocephalella gayi, Am. J. Anat., 131 (1971) 443-462. [13] Taylor, A.C. and Kollros, J.J., Stages in the normal development of Rana pipiens larvae, Anat. Rec., 94 (1946) 7-25. [14] Uga, S. and Hama, K., Electron microscopic study on the synaptic region of the taste organ of carps and frogs, J. Electronmicrosc., 16 (1967) 269-276.