Neuroscience,1977. Vol. L pp. 781-789. Pergamon Rem. Printed in Great Britain.
ULTRASTRUCTURE OF THE EPITHELIAL SENSORY REGION OF THE LIP IN THE SNAIL HELIX POMATIA L. I. BENEDECZKY Biological Research Institute of Hungarian Academy of Sciences, Tihany, Hungary Abstract--The ultrastructural organization of the sensory epithelium in the lip of the snail, Helix pomtiu, has been studied. Both the lateral and ventral surface of the lip are composed of columnar,
unciliated epithelial cells. At the free surface of indifferent epithelial cells many branching cytoplasmic processes were observed overlying a finely granular cuticular layer 1-3 pm thick. Intraepithelial receptor cells were not present, but a great number of sensory dendrites occurred, especiany on the ventral surface.of the lip. Ou the basis of their ultrastructural characteristics, dendrites could be divided into two main groups. Most of the dendrites bear only microvilli on their distal ends (first group) and smaller population (second group) have both microvilli and cilia at their distal end In the nonciliated dendrites, there was a large amount of smooth surfaced endoplasmic reticulum; in the ciliated type of dendrite, microtulules and other cell organelles were common. It can be concluded that there are at least two types of sensory dendrite in the lip of H. porn&u, and this may be the structural basis of the physiologically established sensitivity to different chemical
substances. NEUROMORPHOUX~ICAL studies on the epithelium of molluscs were 6rst described in the second half of the last century (LACA~IZ-DUTHIER~, 1866; EDINGER, 1877; RETZIU~,1892; h&TON, 1920; ABRAHAM,1940). In the lip of the snail, which histologically belongs to the skin, SCHULZ (1938) distinguished four types of receptors. According to Schulz, the first simple receptor, which most closely resembles the epidermal cell, is involved with general chemical reception, while the other three types are specialised mostly for the reception of tastes. On the basis of physiological experiments (KIECKEBUSH, 1953) it has been suggested that receptors of the lip can distinguish between different chemical sub stances. Further physiological experiments (SALANKI & BAY, 1975) established that ‘the chemical sensitivity of the lip receptors to various chemicals is different’. Thus, both the light microscopical findings and the physiological experiments suggest that in the lip of the snail there are different types of receptor cells. Since there do not seem to be any electron microscopical studies concerning the epithelial receptors of the lip of the snail, such an investigation has now been carried out to see whether the light microscopical findings can be corroborated and whether it is possible to identify a morphological basis for the different chemical sensitivities established by physiological methods. The fine structural characteristics and organisation of the lip of the snail, especially the nerve terminals of receptor cells, will be described. MATERIAL AND METHODS Adult snails, collected in spring and autumn, were used in our experiment. Before fixation the shell of the animals was removed and the lip was cut off by sharp scissors. After removal, the lips were cut into 1 mm cubes. A few 781
animals were anesthetized with magnesium chloride (0.5 ml of 10% solution was injected into the lymphatic sack) before cutting off the lips. Several different fixatives were used in our experiment, but only two of them gave satisfactory preservation. The compositions of these fixatives were: (1) Two per cent (w/v) OsO,, in s-Collidine buffer (0.06 M), pH = 7.3; (2) Two per cent (w/v) 0~0~ + 3% (w/v) glutaraldehyde, in sCollidine buffer (0.06 M), pH = 7.3. Fixation in the first fixative was carried out at 4°C for 4h, while for the second fixative the time of prefixation was 2 h followed by another 2 h in fixative No. 1. After fixation blocks were dehydrated in ethanol (30% (v/v), SO%, 75%, 96”/ 100%). Blocks were stained for 1 h in 0.5% (w/v) uranyl acetate dissolved in 75% (v/v) ethanol. Samples were embedded in Durcupan ACM. Ultrathin sections were cut with an LKB ultratome and stained by the lead citrate method of REYNOLDS (1963) for 5 min. Sections were examined in a TESLA BS 413 A electron microscope.
RESULTS Among the different fixatives tested, the best preservation was achieved with No. 2, containing both
0s04 and ghitaraldehyde. Most of the micrographs illustrated were chosen from this fixation. Epithelial cells Under the low power magnification it was found that both the lateral and ventral surface of the lip are composed of different types of whmmar epithelial cells (Fig. 1). On the apical surface (i.e. the free surface) of epithelial cells a rather thick finely granulated cuticular layer was found from which lingerlike long cytoplasmic processes arose. These presented a variety of shapes and sizes. Some were branched and there were bundles of filaments in many. The lateral
781
I. BENEDECZK~
plasma membrane of adjacent epithelial cells are extensively interdigitated, and the intracellular spaces between them often contain electron-dense material (Figs 1, 4). The indifferent epithelial ceils are connected with zonula adherents at the apical poles, under which lie the long segments of septate desmosomes. A large number of different cell organelles (mitochondria, multivesicular bodies, secretion and prgment granules) are present in the electron-dense cytoplasm of the epithelial cells. The Golgi apparatus is rather well developed, and an abundant rough surfaced endoplasmic reticulum is characteristic of the epithelial cells.Besides the ubiquitous organelles, the cells always contain a large amount of electron-dense bundles of long filaments (Figs 1, 6). The nucleus is located at the basal part of the cell (Fig. 6). At the base of the cells there is an electron-dense, 0.1-0.2 grn thick, basament membrane. along which lie a great number of collagen fibrils (Fig. 9). ~e?zLir~tesof receptor fells
The nuclei and perikarya of the subepithelial receptor cells were not observable in the epithelial layer of the lip either in the lateral or in the ventral surface; however. their dendrites were easily detectable between the epithelial cells. The sensory dendrites were more frequently found on the ventral surface of the lip than on the lateral surface, where their number was very low. On the basis of their ultrastructural characteristics two types of sensory dendrites can be distinguished: (1) Free nerve ending with rni~ov~ll~ (2) Free nerve ending with microvilli and cilia. Dendrztes of type 1. The first type of sensory dendrite was more common than the second one in the ventral surface of the lip. The frequency of dendrites among epithelial cells is demonstrated in Fig. 1. In the plane of section after each sixth epithelial cell one dendrite follows. Sometimes the number of dendrites equals the number of epithelial cells (Fig. 3). Nevertheless the abundance of dendrites is not characteristic for the whole surface of the lip so the distribution of dense areas is mosaic-like. On the basis of their fine structural features. the sensory dendrites can easily be drstinguished from the neighbouring nonsensory epithelial cells. With respect to microvilli, those of the sensory dendrites-though highly variable-are fewer, thinner, less regular in outline and much less frequently branched than are those of the regular non-sensory epithelial cells. In addition, electron dense bundles of filaments are absent. Sometimes a localized cytoplasmic protrusion from the apical portion of a dendrite penetrates into or even through the overlying cuticular layer. The morphological appearance of the dendrites is rather variable. There is sometimes a great number of microvilli on the top of the dendrite (Figs 1, 4) but is is also common that one or two microvilli are present only at the distal end of the sensory dendrite (Fig. 5). Dendrites without microvilli also occur (Fig.
3). The electron density of dendritic cytoplasm depends mainly on the mode of fixation. With double fixation. the density of the dendrite is usually the same as that of the epithelial cells (Figs 1, 4). After 0~0, fixation the density of the dendritic cytoplasma is always less than that of the epithelial cells (Figs 2, 3, 5). Sometimes it is possible to trace a fortunate plane in the sections, where the dendrites are parallel with the length of the epithelial cells, so a relatively long part of the dendrite is visible (Figs 2-5). The apical plasma membrane of the dendrite is attached usually with zonula adherens to the membrane of the epithelial cell and, as with the nonsensory cell boundaries, proximal to the zonula adherens, one finds septate desmosomes sealing the intercellular spaces. The dendritic processes of the sensory receptor cells are usually rich in cell organelles; the most conspicuous is the abundant presence of smooth-surfaced endoplasmic reticulum (Figs 24). The largest quantity of endoplasmic reticulum IS concentrated in the distal part of the dendrite. Proximal to the septate desmosomes the volume of endoplasmlc reticulum usually decreases (Fig. 2) but sometimes it occurs in the whole area of the dendrite (Fig. 3). The vesicles of smooth-surfaced endoplasmic reticulum often seem to be attached to the septate desmosomes. or to the inner membrane surface of the dendrite (Fig. 4). With 0~0, fixed material, the vesicles form lines along the whole length of the dendrite (Fig. 5). Relatively few mitochondria and multivesicular bodies were found in the dendritic cytoplasm (Figs 2 and 5). Free ribosomes and micro~laments always occur in the dendrite, usually evenly distributed (Figs 2-S). Microtubules are present mostly in the proximal part of the dendrites (Fig. 2). Dendrites of type 2. Sensory dendrites having both microvilli and cilia are not so common in the lip of the snail as the nonciliated type of dendrite. The esttmation of the number of cilia is rather difficult because it needs serial sections. Mostly we have found more than five cilia in a given plane of section at the top of the dendrite (Figs 6, 7). In a cross-section of a cdium, the typical 9 + 2 tubules were usually present. At the basal part of the cilia one generally finds the dense plaques of the basal bodies (Figs 7, 8) and in a fortunate plane the rootlets also are visible (Fig. 6). Ciliated dendrites usually have a pale neuroplasma (Figs 7--9) and their cell boundaries: zonula adherens and septate desmosomes are organized in a similar way as in the nonciliated dendrites (Figs 7, 8). In contrast to the nonciliated dendrites, the ciliated ones contain a rather large amount of cell organelles (mostly mitochondria and multivesicular bodies) (Figs 6-8) and they are also rich in microtubules. On the other hand there is a smaller amount of smooth-surfaced endoplasmic reticulum in the ciliated dendrites. Sometimes we were able to follow the passage of the dendrite from the basal pole of nonsensory epithelial cells to the apical pole, which corresponds to a distance of about 20,~m (Fig. 6).
Ultrastructure of the sensory region of the lip of snail
783
However ZYLSTRA(1972b) recently demonstrated six types of free nerve endings in his electron microscopic paper, and the seventh is the so called intraepithelial receptor cell. In contrast to this CRISP (1971) and NAVONK (1973) identified only two kinds of free nerve endings in the skin of molluscs. From the results of the above studies it can be concluded that two types of free nerve endings, namely the ciliated and nonciliated ones, undoubtedly occur in the skin of molDISCUSSION lusts. These two basic types of free nerve endings also occur in the lip of H. pomatia. The fact that both The ultrastructural characteristics of epithelial receptor cells have been described in the last 10 years ciliated and nonciliated dendrites are unevenly distributed in the epithelium of the lip, and furthermore for many different species of mollusc. The photoreceptors were extensively studied (BARBER& WRIGHT, that its ventral surface usually contains more den1969; RENZONI, 1968; ROGWS, 1971; STORCEE,1972) drites than the lateral surface, are consistent with the results of physiological experiments (SALANKI & BAY, but several papers have also been published concerning the tentacles of the snail (SCH~ALBACH& LICKE- 1975) where the ventral side of the lip was found to be much more sensitive to chemicals than the lateral FELD, 1962; LANE, 1963; RENZONI, 1968; Ro~naa, 1971; ZYLSTRA,1972a, b; CRISP, 1971; NAVONI, 1973; side. The so-called nonciliated group of free nerve endings, although the commonest type, was not comWRIGHT, 1974u, b; WONDRAK,1975). Detailed descrippletely homogeneous, since the number, size and tions have been given of the different epithelial receptors for two species by CRISP (1971) and ZYL~TRA lengths of the microvilli can be very different. It is, of course, possible that some of these apparent mor(197242,b). phological differences may derive from the plane of Although there are some ultrastructural observations concerning the receptor cells in the sucker of section, rather than having any functional signifithe octopus, as well as on the lip of the squid (GRA- cance. In relation to the two types of sensory dendrite ZUDEI, 1960, 1%5; EMERY, 197&b) very little is the question arises, what type of reception does each known about the fine structural or~ni~tion of mediate? According to CRISP (1971) the ciliated nerve receptor cells in the mouth region of molluscs. endings are capable of both chemo- and mechanoAs regards receptor cells in general, most authors reception. On the other hand ZYLFXRA (1972b) sugstate that, irrespective of the localization of receptors, gests that nonciliated dendrites of receptor cells are nerve cells are the primary sense cells in the skin also capable of chemoreception. NAVONI (1973) epithelium. Only STORCH(1972) believed that secondbelieves that ciliated sensory cells are responsible for ary sense cells also occur in some Prosobranchia. Our chemoreception while the nonciliated free nerve endobservations also suggest that the dendrites frequently found in the lip of the snail were the processes of ings are involved in the perception of tactile stimuli. the primary sense cells, which lie deep under the skin These examples show that at present it is impossible to determine exactly what kind of reception is perepithelium, probably in a ganglion. formed by a given mo~hologi~l type of sensory endIt is not surprising that the ultrastructural organization of the primary receptor cells in molluscs is ings. The large amount of the smooth vesicles and far from uniform, because studies have been made tubules in the apical part of nonciliated dendrites is on very different species. Differences might, however, noteworthy. Ultrastructurally this appears to be also derive from the method of fixation. Since the smooth surfaced endoplasmic reticulum. The fact that preservation of the tissue was often unsatisfactory, it occurs mostly in the nonciliated dendrites, gives authors have had to try a range of different fixatives. an opportunity for the more exact classification of In the present study, the simple epithelial cells first this type of dendrite. It is well known that in the of all had to be differentiated from nerve elements. This is ~portant because in the skin of mollusa it dendrites of vertebrates the endoplasmic reticulum occurs both in ovulated and agramtlated forms; is easy to find ciliated epithelial cells having no rempnevertheless smooth surfaced endoplasmic reticulum tar function (CRISP, 1971). Thus not all the ciliated is quite rare in the dendrites of invertebrate molluscs cells are necessarily sensory cells. (WONDRAK,1975). However, AROS& VIGH (1971) dePossible structural-function correlation scribed a similar complicated tubular system in the It is a common structural feature of most primary dendritic processes of the skin of the earthworm. receptor cells that the cell body is deeply embedded These authors supposed that the tubules might oriunder the epithelial cells, thus intraepithelial receptor ginate in the plasma membrane or might belong to cells are quite rare (ZYLSTRA,1972~). There are’several the endoplasmic reticulum, and speculated that they types of morphologically distinct skin receptors in might increase the surface area of the plasma memmolluscs. In the light microscopic study of SCXILZ brane. There is no doubt that these structures are (1938) only four types of receptor cells are described. often in close contact with the plasma membrane also At the basal part of the epithelial cells (Fig. 9) the bundles of dendrites can be seen to break through the basal lamina and go forward to the apex of the epithelial cells. The proximal parts of the dendrites are rich in microtubules, but poor in other cell organelles. Under the epithelial cells there is a thin layer of the basal membrane to which a large mount of collagen fibrils are attached (Fig. 9).
784
FIG. 1. Low power electron micrograph of the hp of Helix pomatuz Indifferent epidermal cells (E) are easily detectable on the basis of long ramifications of their cytoplasmic processes and on the extremely interdigitated plasma membrane (pm). A sensory dendrite (D) with short microvilli also occurs among the epidermal cells. x 12,000. FIG. 2. Dendrite (D) of receptor cell from the ventral surface of the lip. The cytoplasm of the epidermal cells (E) is rather electron dense at the same time that of the sensory dendrite is light. At the level of cell junctions (CJ) a large amount of smooth-surfaced endoplasmic reticulum (sEr) occurs m the dendrite, below this microtubules (MT) and mitochondria (M) are present in the light zone. x 24,000. FIG. 3. Sometimes the sensory dendrites (D) are quite frequent among epithelial cells (E). In this case the ending of the sensory cell looks like a knob, and its cytoplasm contains a large amount of smooth-surfaced endoplasmic reticulum (sEr). x 24,000. FIG. 4. Distal end of a sensory dendrite (D) in the lip of Helix pomatia. At the top of the dendrite a number of microvilli (Mv) can be seen. Zonula adherens (ZA) and septate desmosomes (SD) join the dendrite to the neighbouring epithelial cells (E). The smooth-surfaced endoplasmic reticulum (sEr) IS attached mainly to the membrane of the dendrite. Note the presence of free ribosomes (R) in the dendrite. x 30,000. FIG. 5. The neuroplasm of the sensory dendrites (D) is usually lighter than the cytoplasm of the epithelial cells (E) in 0~0, fixed material. At the top of the dendrite there is only one microvillus (Mv). The dendritic cytoplasm contains lines of vesicles (V). Note mitochondna (M) and multivesicular body in the dendrite. x 30,000. FIG. 6. Ciliated sensory dendrite (DC) from the ventral surface of the lip. The dendrite is visible from the basal part of epithelial cells (E) to the apical pole. The dendritic cytoplasm is rather light, mitochondria (M), microtubules (MT), and multivesicular body (MB) occur in it. At the top of the dendrite 4 cilia (C) are present. Well-developed Golgi apparatus (G) is characteristic of the epithehal cells (E). Note the nucleus and rootless (R) in the cells. x 12,000. FIG. 7. Apical pole of a ciliated dendrite (DC). A large number of cilia (C) can be seen at the top of this dendrite. In a cross-section the typical 9 + 2 tubules are detectable in the cihum. Among the cilia, microvilli (Mv) also occur. In some cases the basal body of the cilia is also clearly detectable, while the rootlets were not observable. The dendritic cytoplasm is very transparent, the number of cell organelles is rather low. Note the presence of zonula adherens (ZA) on the cell membrane. x 16,000. FIG. 8. Oblique section of a ciliated sensory dendrite (DC). The dentritic cytoplasm is poor in cell organelles, only the basal bodies of the cilia (Bb), some mitochondria (M) and a few vesicles (V) are present. The zonula adherens (ZA) and the septums of the septate desmosomes (SD) are clearly seen. In the neighbouring epithelial cells (E) the bundles of filaments (F) are conspicuous. x 42,000. FIG. 9. Subepithelial layer of the lip. Branches of sensory dendrites (D) break through the basal lamina (BL) of epithelial cells (E) and reach the surface of the epidermis. The proximal part of the dendrites (D) usually contains a great number of microtubules (MT); moreover mitochondria (M) as well as some vesicles (V) also occur in the dendritic cytoplasm. Abundant collagen (CO) is present in the interstitium (I). x 16,ooO.
785
FIGS 1, 2, and 3.
786
FIGS 4 and 5.
787
FIGS6 and 7.
788
FIGS 8 and 9.
Ultrastructure
of the sensory region of the lip of snail
in the snail, but this contact alone is not satisfactory proof that they originated from the plasma membrane. Just as we do not know the exact origin of this structure, we do not know what its function is in the dendrites. It is possible that, since the smooth
789
surface endoplasmic reticulum has an important role in vertebrate 41 metabolism, the dendritic smoothsurfaced tubules may pIay a role in the metabolism of the invertebrate nerve celI and might also be involved in detoxification processes.
REFERENCES A. (1940) Die Innervation des Darmkanals der Gastropoden. Z. Zellforsch. mikrosk. Anat., A 30, 273-296. Amos B. P. & VIC~HB. (1971) Fine structure of the peripheral sensdry cells in the earthworm De~ro~~nu octuedra. Acta biot. hung. 22, 443-456. BARBERV. & WRIGHT D. E. (lW9) The fine structure of the eye and optic tentacle of the mollusc Cardium edule. J. Ultrastnrct. Res. 26, 515-528. CRISP M. (1971) Structure and abundance of receptors of the unspecialized external epithelium of Nassurius reticulatus (Gastropoda, ~osobr~chia~. J. mar. biol. Ass., U.K. 51, 865-890. EDIWER L. (1877) Die Endigung der Hautnerven bei Pterotrachea. Arch. mikrosk. Anat. EntwMech. 14, 171-179. E~Y D. G. (1975a) Ciliated sensory neurons in the lip of the squid Lolliguncula brevis blainuille. Cell 7’iss. Res. 157, 323-329. E~IERYD. G. (1975b) Ciliated sensory cells and associated neurons in the lip of Octopus joubini robson. Cell Tiss. Res. 157, 331-340. GRAZIADEIP. P. C. (1960) Primi dati sul corredo nervoso de1 labbro oraie di Sepia o@ianulis. Acad. Naz. Lincei 29, 398-400. GRAZIADEIP. P. C. (1965) Sensory receptor cells and related neurons in cephalopods. Cold Spring Harb. Symp. quant. Biol. 30, 45-57. KIEYXEBUSCHW. (1953) Beitrag zur Physiologie des cbemischen Sines von Helix pomatia. Zool. Jb (Physiol.) 64, 154-182. LACAZE-DUTHIERS H. (1866) On the multiplicity and termination of the nerves in the Mollusca. Ann. Mag. nat. Hist. 17, 157-159. LANE N, J. (1963) Microvilli on the external surfaces of gastropod tentacles and body walls. Q. JZ microsc. Sci. 104, 495-504. MERTONH. (1920) Untersuchungen iiber die Hautsinnersorgane der Mollusken-I. Opistobranchia. Abh. senckenb. naturforsch. Ges. 36, 447-473. NAVONI L. (1973) Der chemische Sinn der ~udibr~chier (Gastropoda, Op~tobr~c~a). Rev. suisse Zool. 79, 1333-1379. RENZONIA. (1968) Observazioni istologiche, i&chin&he ed ultrastrutturali sui tentacoli di Vaginulusborellionus.Gastrdpoda. Soleolifera. Z. Zellforsch. mikrosk. Anat. 87, 350-376. RETZNS G. (1892) Das sensible Nervensystem der Mollusken. Biol. Unters. N.F. 4, 11-18. REYNOLDSE. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. cell Biol. 17, 208-212. ROCERSD. C. (1971) Surface specializations of the epithelial cells at the optic tentacle, dorsal surface of the head and ventral surface of the foot in Helix aspersa. Z. Zellforseh. mikrosk. Anat. 114, 106-l 16. SAL&WI J. & TRUONG VAN BAY (1975) Sensory input characteristics at the chemical stimulation of the lip in the snail Helix posts L. Annal. Biol. ~ih~y 42, 115-128. SCHULZF. (1938) Bau und Funktion der Sinneszellen in der K&peroberf&he von Helix pomatia. 2. Morph. fikol. Tiere 33, 555-581. SCHWALBACHG. & LICKEFELDK. (1962) Die Epidermis Morphologie der Sinneskalotte von Helix pomutia L. Z. Zellforsch. mikrosk. Anat. 58, 277-288. STARCHV. (1972) Elek~on~~~o~opi~he und hist~e~~he Unt~such~g~ llber Rezeptoren von Gastropoden (Prosobranchia, Opistobranchia. Z. wiss. Zool. 184, l-26. WONDRAKG. (1975) The ultrastructure of the sensory cells in the chemoreceptor of the ommatophore of Helix pamatia L. Cell Tiss. Res. 159, 121-140. WRIGHTB. R. (1974a) Sensory structure of the tentacles of a slug, Arion ater (Pulmonata, Molluscat_l. The ultrastructure of the distal epithelium, receptor cells and tentacular ganglion. Cell Tiss. Res. 151, 229-244. WRIGHT B. R. (1974b) Sensory structure of the tentacles of the slug, Arian ater (Pulmonata, Mollusca). Cell. Tiss. Res. 151, 245-257. ZYLSTRAU. (1972a) Hi&chemistry and ultrastructure of the epidermis and the subepidermal @and cells of the freshwater snails Lymnoea s~~lis and B~rn~~~ pfei~ri. Z. Zellfor~h. ~~0~. Anot. 130, 93-134. ZWTRA U. (19726) Distribution and ultrastrncture of epidermal sensory cells in the freshwater snails Lymnaea stagnalis and Biomphalariapfeifferi. Neth. J. Zool. 22, 283-298. ABRAHAM
(Accepted 14 April 1977)
ULTRASTRUCTURE OF THE EPITHELIAL SENSORY REGION OF THE LIP IN THE SNAIL HELIX POMATIA L. I. BENEDECZKY Biological Research Institute of Hungarian Academy of Sciences, Tihany, Hungary Abstract--The ultrastructural organization of the sensory epithelium in the lip of the snail, Helix pomtiu, has been studied. Both the lateral and ventral surface of the lip are composed of columnar,
unciliated epithelial cells. At the free surface of indifferent epithelial cells many branching cytoplasmic processes were observed overlying a finely granular cuticular layer 1-3 pm thick. Intraepithelial receptor cells were not present, but a great number of sensory dendrites occurred, especiany on the ventral surface.of the lip. Ou the basis of their ultrastructural characteristics, dendrites could be divided into two main groups. Most of the dendrites bear only microvilli on their distal ends (first group) and smaller population (second group) have both microvilli and cilia at their distal end In the nonciliated dendrites, there was a large amount of smooth surfaced endoplasmic reticulum; in the ciliated type of dendrite, microtulules and other cell organelles were common. It can be concluded that there are at least two types of sensory dendrite in the lip of H. porn&u, and this may be the structural basis of the physiologically established sensitivity to different chemical
substances. NEUROMORPHOUX~ICAL studies on the epithelium of molluscs were 6rst described in the second half of the last century (LACA~IZ-DUTHIER~, 1866; EDINGER, 1877; RETZIU~,1892; h&TON, 1920; ABRAHAM,1940). In the lip of the snail, which histologically belongs to the skin, SCHULZ (1938) distinguished four types of receptors. According to Schulz, the first simple receptor, which most closely resembles the epidermal cell, is involved with general chemical reception, while the other three types are specialised mostly for the reception of tastes. On the basis of physiological experiments (KIECKEBUSH, 1953) it has been suggested that receptors of the lip can distinguish between different chemical sub stances. Further physiological experiments (SALANKI & BAY, 1975) established that ‘the chemical sensitivity of the lip receptors to various chemicals is different’. Thus, both the light microscopical findings and the physiological experiments suggest that in the lip of the snail there are different types of receptor cells. Since there do not seem to be any electron microscopical studies concerning the epithelial receptors of the lip of the snail, such an investigation has now been carried out to see whether the light microscopical findings can be corroborated and whether it is possible to identify a morphological basis for the different chemical sensitivities established by physiological methods. The fine structural characteristics and organisation of the lip of the snail, especially the nerve terminals of receptor cells, will be described. MATERIAL AND METHODS Adult snails, collected in spring and autumn, were used in our experiment. Before fixation the shell of the animals was removed and the lip was cut off by sharp scissors. After removal, the lips were cut into 1 mm cubes. A few 781
animals were anesthetized with magnesium chloride (0.5 ml of 10% solution was injected into the lymphatic sack) before cutting off the lips. Several different fixatives were used in our experiment, but only two of them gave satisfactory preservation. The compositions of these fixatives were: (1) Two per cent (w/v) OsO,, in s-Collidine buffer (0.06 M), pH = 7.3; (2) Two per cent (w/v) 0~0~ + 3% (w/v) glutaraldehyde, in sCollidine buffer (0.06 M), pH = 7.3. Fixation in the first fixative was carried out at 4°C for 4h, while for the second fixative the time of prefixation was 2 h followed by another 2 h in fixative No. 1. After fixation blocks were dehydrated in ethanol (30% (v/v), SO%, 75%, 96”/ 100%). Blocks were stained for 1 h in 0.5% (w/v) uranyl acetate dissolved in 75% (v/v) ethanol. Samples were embedded in Durcupan ACM. Ultrathin sections were cut with an LKB ultratome and stained by the lead citrate method of REYNOLDS (1963) for 5 min. Sections were examined in a TESLA BS 413 A electron microscope.
RESULTS Among the different fixatives tested, the best preservation was achieved with No. 2, containing both
0s04 and ghitaraldehyde. Most of the micrographs illustrated were chosen from this fixation. Epithelial cells Under the low power magnification it was found that both the lateral and ventral surface of the lip are composed of different types of whmmar epithelial cells (Fig. 1). On the apical surface (i.e. the free surface) of epithelial cells a rather thick finely granulated cuticular layer was found from which lingerlike long cytoplasmic processes arose. These presented a variety of shapes and sizes. Some were branched and there were bundles of filaments in many. The lateral
781
I. BENEDECZK~
plasma membrane of adjacent epithelial cells are extensively interdigitated, and the intracellular spaces between them often contain electron-dense material (Figs 1, 4). The indifferent epithelial ceils are connected with zonula adherents at the apical poles, under which lie the long segments of septate desmosomes. A large number of different cell organelles (mitochondria, multivesicular bodies, secretion and prgment granules) are present in the electron-dense cytoplasm of the epithelial cells. The Golgi apparatus is rather well developed, and an abundant rough surfaced endoplasmic reticulum is characteristic of the epithelial cells.Besides the ubiquitous organelles, the cells always contain a large amount of electron-dense bundles of long filaments (Figs 1, 6). The nucleus is located at the basal part of the cell (Fig. 6). At the base of the cells there is an electron-dense, 0.1-0.2 grn thick, basament membrane. along which lie a great number of collagen fibrils (Fig. 9). ~e?zLir~tesof receptor fells
The nuclei and perikarya of the subepithelial receptor cells were not observable in the epithelial layer of the lip either in the lateral or in the ventral surface; however. their dendrites were easily detectable between the epithelial cells. The sensory dendrites were more frequently found on the ventral surface of the lip than on the lateral surface, where their number was very low. On the basis of their ultrastructural characteristics two types of sensory dendrites can be distinguished: (1) Free nerve ending with rni~ov~ll~ (2) Free nerve ending with microvilli and cilia. Dendrztes of type 1. The first type of sensory dendrite was more common than the second one in the ventral surface of the lip. The frequency of dendrites among epithelial cells is demonstrated in Fig. 1. In the plane of section after each sixth epithelial cell one dendrite follows. Sometimes the number of dendrites equals the number of epithelial cells (Fig. 3). Nevertheless the abundance of dendrites is not characteristic for the whole surface of the lip so the distribution of dense areas is mosaic-like. On the basis of their fine structural features. the sensory dendrites can easily be drstinguished from the neighbouring nonsensory epithelial cells. With respect to microvilli, those of the sensory dendrites-though highly variable-are fewer, thinner, less regular in outline and much less frequently branched than are those of the regular non-sensory epithelial cells. In addition, electron dense bundles of filaments are absent. Sometimes a localized cytoplasmic protrusion from the apical portion of a dendrite penetrates into or even through the overlying cuticular layer. The morphological appearance of the dendrites is rather variable. There is sometimes a great number of microvilli on the top of the dendrite (Figs 1, 4) but is is also common that one or two microvilli are present only at the distal end of the sensory dendrite (Fig. 5). Dendrites without microvilli also occur (Fig.
3). The electron density of dendritic cytoplasm depends mainly on the mode of fixation. With double fixation. the density of the dendrite is usually the same as that of the epithelial cells (Figs 1, 4). After 0~0, fixation the density of the dendritic cytoplasma is always less than that of the epithelial cells (Figs 2, 3, 5). Sometimes it is possible to trace a fortunate plane in the sections, where the dendrites are parallel with the length of the epithelial cells, so a relatively long part of the dendrite is visible (Figs 2-5). The apical plasma membrane of the dendrite is attached usually with zonula adherens to the membrane of the epithelial cell and, as with the nonsensory cell boundaries, proximal to the zonula adherens, one finds septate desmosomes sealing the intercellular spaces. The dendritic processes of the sensory receptor cells are usually rich in cell organelles; the most conspicuous is the abundant presence of smooth-surfaced endoplasmic reticulum (Figs 24). The largest quantity of endoplasmic reticulum IS concentrated in the distal part of the dendrite. Proximal to the septate desmosomes the volume of endoplasmlc reticulum usually decreases (Fig. 2) but sometimes it occurs in the whole area of the dendrite (Fig. 3). The vesicles of smooth-surfaced endoplasmic reticulum often seem to be attached to the septate desmosomes. or to the inner membrane surface of the dendrite (Fig. 4). With 0~0, fixed material, the vesicles form lines along the whole length of the dendrite (Fig. 5). Relatively few mitochondria and multivesicular bodies were found in the dendritic cytoplasm (Figs 2 and 5). Free ribosomes and micro~laments always occur in the dendrite, usually evenly distributed (Figs 2-S). Microtubules are present mostly in the proximal part of the dendrites (Fig. 2). Dendrites of type 2. Sensory dendrites having both microvilli and cilia are not so common in the lip of the snail as the nonciliated type of dendrite. The esttmation of the number of cilia is rather difficult because it needs serial sections. Mostly we have found more than five cilia in a given plane of section at the top of the dendrite (Figs 6, 7). In a cross-section of a cdium, the typical 9 + 2 tubules were usually present. At the basal part of the cilia one generally finds the dense plaques of the basal bodies (Figs 7, 8) and in a fortunate plane the rootlets also are visible (Fig. 6). Ciliated dendrites usually have a pale neuroplasma (Figs 7--9) and their cell boundaries: zonula adherens and septate desmosomes are organized in a similar way as in the nonciliated dendrites (Figs 7, 8). In contrast to the nonciliated dendrites, the ciliated ones contain a rather large amount of cell organelles (mostly mitochondria and multivesicular bodies) (Figs 6-8) and they are also rich in microtubules. On the other hand there is a smaller amount of smooth-surfaced endoplasmic reticulum in the ciliated dendrites. Sometimes we were able to follow the passage of the dendrite from the basal pole of nonsensory epithelial cells to the apical pole, which corresponds to a distance of about 20,~m (Fig. 6).
Ultrastructure of the sensory region of the lip of snail
783
However ZYLSTRA(1972b) recently demonstrated six types of free nerve endings in his electron microscopic paper, and the seventh is the so called intraepithelial receptor cell. In contrast to this CRISP (1971) and NAVONK (1973) identified only two kinds of free nerve endings in the skin of molluscs. From the results of the above studies it can be concluded that two types of free nerve endings, namely the ciliated and nonciliated ones, undoubtedly occur in the skin of molDISCUSSION lusts. These two basic types of free nerve endings also occur in the lip of H. pomatia. The fact that both The ultrastructural characteristics of epithelial receptor cells have been described in the last 10 years ciliated and nonciliated dendrites are unevenly distributed in the epithelium of the lip, and furthermore for many different species of mollusc. The photoreceptors were extensively studied (BARBER& WRIGHT, that its ventral surface usually contains more den1969; RENZONI, 1968; ROGWS, 1971; STORCEE,1972) drites than the lateral surface, are consistent with the results of physiological experiments (SALANKI & BAY, but several papers have also been published concerning the tentacles of the snail (SCH~ALBACH& LICKE- 1975) where the ventral side of the lip was found to be much more sensitive to chemicals than the lateral FELD, 1962; LANE, 1963; RENZONI, 1968; Ro~naa, 1971; ZYLSTRA,1972a, b; CRISP, 1971; NAVONI, 1973; side. The so-called nonciliated group of free nerve endings, although the commonest type, was not comWRIGHT, 1974u, b; WONDRAK,1975). Detailed descrippletely homogeneous, since the number, size and tions have been given of the different epithelial receptors for two species by CRISP (1971) and ZYL~TRA lengths of the microvilli can be very different. It is, of course, possible that some of these apparent mor(197242,b). phological differences may derive from the plane of Although there are some ultrastructural observations concerning the receptor cells in the sucker of section, rather than having any functional signifithe octopus, as well as on the lip of the squid (GRA- cance. In relation to the two types of sensory dendrite ZUDEI, 1960, 1%5; EMERY, 197&b) very little is the question arises, what type of reception does each known about the fine structural or~ni~tion of mediate? According to CRISP (1971) the ciliated nerve receptor cells in the mouth region of molluscs. endings are capable of both chemo- and mechanoAs regards receptor cells in general, most authors reception. On the other hand ZYLFXRA (1972b) sugstate that, irrespective of the localization of receptors, gests that nonciliated dendrites of receptor cells are nerve cells are the primary sense cells in the skin also capable of chemoreception. NAVONI (1973) epithelium. Only STORCH(1972) believed that secondbelieves that ciliated sensory cells are responsible for ary sense cells also occur in some Prosobranchia. Our chemoreception while the nonciliated free nerve endobservations also suggest that the dendrites frequently found in the lip of the snail were the processes of ings are involved in the perception of tactile stimuli. the primary sense cells, which lie deep under the skin These examples show that at present it is impossible to determine exactly what kind of reception is perepithelium, probably in a ganglion. formed by a given mo~hologi~l type of sensory endIt is not surprising that the ultrastructural organization of the primary receptor cells in molluscs is ings. The large amount of the smooth vesicles and far from uniform, because studies have been made tubules in the apical part of nonciliated dendrites is on very different species. Differences might, however, noteworthy. Ultrastructurally this appears to be also derive from the method of fixation. Since the smooth surfaced endoplasmic reticulum. The fact that preservation of the tissue was often unsatisfactory, it occurs mostly in the nonciliated dendrites, gives authors have had to try a range of different fixatives. an opportunity for the more exact classification of In the present study, the simple epithelial cells first this type of dendrite. It is well known that in the of all had to be differentiated from nerve elements. This is ~portant because in the skin of mollusa it dendrites of vertebrates the endoplasmic reticulum occurs both in ovulated and agramtlated forms; is easy to find ciliated epithelial cells having no rempnevertheless smooth surfaced endoplasmic reticulum tar function (CRISP, 1971). Thus not all the ciliated is quite rare in the dendrites of invertebrate molluscs cells are necessarily sensory cells. (WONDRAK,1975). However, AROS& VIGH (1971) dePossible structural-function correlation scribed a similar complicated tubular system in the It is a common structural feature of most primary dendritic processes of the skin of the earthworm. receptor cells that the cell body is deeply embedded These authors supposed that the tubules might oriunder the epithelial cells, thus intraepithelial receptor ginate in the plasma membrane or might belong to cells are quite rare (ZYLSTRA,1972~). There are’several the endoplasmic reticulum, and speculated that they types of morphologically distinct skin receptors in might increase the surface area of the plasma memmolluscs. In the light microscopic study of SCXILZ brane. There is no doubt that these structures are (1938) only four types of receptor cells are described. often in close contact with the plasma membrane also At the basal part of the epithelial cells (Fig. 9) the bundles of dendrites can be seen to break through the basal lamina and go forward to the apex of the epithelial cells. The proximal parts of the dendrites are rich in microtubules, but poor in other cell organelles. Under the epithelial cells there is a thin layer of the basal membrane to which a large mount of collagen fibrils are attached (Fig. 9).
784
FIG. 1. Low power electron micrograph of the hp of Helix pomatuz Indifferent epidermal cells (E) are easily detectable on the basis of long ramifications of their cytoplasmic processes and on the extremely interdigitated plasma membrane (pm). A sensory dendrite (D) with short microvilli also occurs among the epidermal cells. x 12,000. FIG. 2. Dendrite (D) of receptor cell from the ventral surface of the lip. The cytoplasm of the epidermal cells (E) is rather electron dense at the same time that of the sensory dendrite is light. At the level of cell junctions (CJ) a large amount of smooth-surfaced endoplasmic reticulum (sEr) occurs m the dendrite, below this microtubules (MT) and mitochondria (M) are present in the light zone. x 24,000. FIG. 3. Sometimes the sensory dendrites (D) are quite frequent among epithelial cells (E). In this case the ending of the sensory cell looks like a knob, and its cytoplasm contains a large amount of smooth-surfaced endoplasmic reticulum (sEr). x 24,000. FIG. 4. Distal end of a sensory dendrite (D) in the lip of Helix pomatia. At the top of the dendrite a number of microvilli (Mv) can be seen. Zonula adherens (ZA) and septate desmosomes (SD) join the dendrite to the neighbouring epithelial cells (E). The smooth-surfaced endoplasmic reticulum (sEr) IS attached mainly to the membrane of the dendrite. Note the presence of free ribosomes (R) in the dendrite. x 30,000. FIG. 5. The neuroplasm of the sensory dendrites (D) is usually lighter than the cytoplasm of the epithelial cells (E) in 0~0, fixed material. At the top of the dendrite there is only one microvillus (Mv). The dendritic cytoplasm contains lines of vesicles (V). Note mitochondna (M) and multivesicular body in the dendrite. x 30,000. FIG. 6. Ciliated sensory dendrite (DC) from the ventral surface of the lip. The dendrite is visible from the basal part of epithelial cells (E) to the apical pole. The dendritic cytoplasm is rather light, mitochondria (M), microtubules (MT), and multivesicular body (MB) occur in it. At the top of the dendrite 4 cilia (C) are present. Well-developed Golgi apparatus (G) is characteristic of the epithehal cells (E). Note the nucleus and rootless (R) in the cells. x 12,000. FIG. 7. Apical pole of a ciliated dendrite (DC). A large number of cilia (C) can be seen at the top of this dendrite. In a cross-section the typical 9 + 2 tubules are detectable in the cihum. Among the cilia, microvilli (Mv) also occur. In some cases the basal body of the cilia is also clearly detectable, while the rootlets were not observable. The dendritic cytoplasm is very transparent, the number of cell organelles is rather low. Note the presence of zonula adherens (ZA) on the cell membrane. x 16,000. FIG. 8. Oblique section of a ciliated sensory dendrite (DC). The dentritic cytoplasm is poor in cell organelles, only the basal bodies of the cilia (Bb), some mitochondria (M) and a few vesicles (V) are present. The zonula adherens (ZA) and the septums of the septate desmosomes (SD) are clearly seen. In the neighbouring epithelial cells (E) the bundles of filaments (F) are conspicuous. x 42,000. FIG. 9. Subepithelial layer of the lip. Branches of sensory dendrites (D) break through the basal lamina (BL) of epithelial cells (E) and reach the surface of the epidermis. The proximal part of the dendrites (D) usually contains a great number of microtubules (MT); moreover mitochondria (M) as well as some vesicles (V) also occur in the dendritic cytoplasm. Abundant collagen (CO) is present in the interstitium (I). x 16,ooO.
785
FIGS 1, 2, and 3.
786
FIGS 4 and 5.
787
FIGS6 and 7.
788
FIGS 8 and 9.
Ultrastructure
of the sensory region of the lip of snail
in the snail, but this contact alone is not satisfactory proof that they originated from the plasma membrane. Just as we do not know the exact origin of this structure, we do not know what its function is in the dendrites. It is possible that, since the smooth
789
surface endoplasmic reticulum has an important role in vertebrate 41 metabolism, the dendritic smoothsurfaced tubules may pIay a role in the metabolism of the invertebrate nerve celI and might also be involved in detoxification processes.
REFERENCES A. (1940) Die Innervation des Darmkanals der Gastropoden. Z. Zellforsch. mikrosk. Anat., A 30, 273-296. Amos B. P. & VIC~HB. (1971) Fine structure of the peripheral sensdry cells in the earthworm De~ro~~nu octuedra. Acta biot. hung. 22, 443-456. BARBERV. & WRIGHT D. E. (lW9) The fine structure of the eye and optic tentacle of the mollusc Cardium edule. J. Ultrastnrct. Res. 26, 515-528. CRISP M. (1971) Structure and abundance of receptors of the unspecialized external epithelium of Nassurius reticulatus (Gastropoda, ~osobr~chia~. J. mar. biol. Ass., U.K. 51, 865-890. EDIWER L. (1877) Die Endigung der Hautnerven bei Pterotrachea. Arch. mikrosk. Anat. EntwMech. 14, 171-179. E~Y D. G. (1975a) Ciliated sensory neurons in the lip of the squid Lolliguncula brevis blainuille. Cell 7’iss. Res. 157, 323-329. E~IERYD. G. (1975b) Ciliated sensory cells and associated neurons in the lip of Octopus joubini robson. Cell Tiss. Res. 157, 331-340. GRAZIADEIP. P. C. (1960) Primi dati sul corredo nervoso de1 labbro oraie di Sepia o@ianulis. Acad. Naz. Lincei 29, 398-400. GRAZIADEIP. P. C. (1965) Sensory receptor cells and related neurons in cephalopods. Cold Spring Harb. Symp. quant. Biol. 30, 45-57. KIEYXEBUSCHW. (1953) Beitrag zur Physiologie des cbemischen Sines von Helix pomatia. Zool. Jb (Physiol.) 64, 154-182. LACAZE-DUTHIERS H. (1866) On the multiplicity and termination of the nerves in the Mollusca. Ann. Mag. nat. Hist. 17, 157-159. LANE N, J. (1963) Microvilli on the external surfaces of gastropod tentacles and body walls. Q. JZ microsc. Sci. 104, 495-504. MERTONH. (1920) Untersuchungen iiber die Hautsinnersorgane der Mollusken-I. Opistobranchia. Abh. senckenb. naturforsch. Ges. 36, 447-473. NAVONI L. (1973) Der chemische Sinn der ~udibr~chier (Gastropoda, Op~tobr~c~a). Rev. suisse Zool. 79, 1333-1379. RENZONIA. (1968) Observazioni istologiche, i&chin&he ed ultrastrutturali sui tentacoli di Vaginulusborellionus.Gastrdpoda. Soleolifera. Z. Zellforsch. mikrosk. Anat. 87, 350-376. RETZNS G. (1892) Das sensible Nervensystem der Mollusken. Biol. Unters. N.F. 4, 11-18. REYNOLDSE. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. cell Biol. 17, 208-212. ROCERSD. C. (1971) Surface specializations of the epithelial cells at the optic tentacle, dorsal surface of the head and ventral surface of the foot in Helix aspersa. Z. Zellforseh. mikrosk. Anat. 114, 106-l 16. SAL&WI J. & TRUONG VAN BAY (1975) Sensory input characteristics at the chemical stimulation of the lip in the snail Helix posts L. Annal. Biol. ~ih~y 42, 115-128. SCHULZF. (1938) Bau und Funktion der Sinneszellen in der K&peroberf&he von Helix pomatia. 2. Morph. fikol. Tiere 33, 555-581. SCHWALBACHG. & LICKEFELDK. (1962) Die Epidermis Morphologie der Sinneskalotte von Helix pomutia L. Z. Zellforsch. mikrosk. Anat. 58, 277-288. STARCHV. (1972) Elek~on~~~o~opi~he und hist~e~~he Unt~such~g~ llber Rezeptoren von Gastropoden (Prosobranchia, Opistobranchia. Z. wiss. Zool. 184, l-26. WONDRAKG. (1975) The ultrastructure of the sensory cells in the chemoreceptor of the ommatophore of Helix pamatia L. Cell Tiss. Res. 159, 121-140. WRIGHTB. R. (1974a) Sensory structure of the tentacles of a slug, Arion ater (Pulmonata, Molluscat_l. The ultrastructure of the distal epithelium, receptor cells and tentacular ganglion. Cell Tiss. Res. 151, 229-244. WRIGHT B. R. (1974b) Sensory structure of the tentacles of the slug, Arian ater (Pulmonata, Mollusca). Cell. Tiss. Res. 151, 245-257. ZYLSTRAU. (1972a) Hi&chemistry and ultrastructure of the epidermis and the subepidermal @and cells of the freshwater snails Lymnoea s~~lis and B~rn~~~ pfei~ri. Z. Zellfor~h. ~~0~. Anot. 130, 93-134. ZWTRA U. (19726) Distribution and ultrastrncture of epidermal sensory cells in the freshwater snails Lymnaea stagnalis and Biomphalariapfeifferi. Neth. J. Zool. 22, 283-298. ABRAHAM
(Accepted 14 April 1977)
