35
The fine structure of certain secretory cells in the optic
tentacles of the snail, Helix aspersa
By NANCY J. LANE
(From the Cytological Laboratory, Department of Zoology, University Museum, Oxford.
Present address: Department of Pathology, Albert Einstein College of Medicine, Yeshiva
University, New York 61, N.Y., U.S.A.)
With 4 plates (figs, i to 4)
Summary
In Helix aspersa, the cytoplasm of the collar cells that surround the tentacular ganglion
contains spheroidal granules of low electron density (a-bodies), and ^3-bodies that
contain electron-dense vesicles within the size range of elementary neurosecretory
granules. Mitochondria and lamellar Golgi complexes are also present. Mitochondria,
a-bodies, fibrils, objects resembling multivesicular bodies, and moderately electrondense granules are found in the lengthy processes into which the collar cells are drawn
out.
One process of each bipolar collar cell seems to merge into the tentacular ganglion.
This consists of neurones with processes that contain mitochondria, vesicles of various
sizes, filaments, and electron-dense granules similar to elementary neurosecretory
granules.
The lateral cells that line the dermo-muscular sheath encasing the optic tentacles
contain in their cytoplasm an endoplasmic reticulum with many ribosomes, mitochondria, Golgi complexes, and many electron-lucent globules. These globules seem
to be elaborated by the lamellar Golgi bodies. There are also electron-dense inclusions, which may be lysosomes, that are scattered sparsely between the other
globules and cytoplasmic components. All these structural details are similar to those
found in some mucus-secreting cells.
The possible nature and origin of the tentacular secretory cells are discussed.
Introduction
T H E optic tentacles of stylommatophoran gastropods contain a number of
cells, of which some appear to be nerve-cells yet possess a great many
spheroidal cytoplasmic inclusions. This combination of characteristics, now
considered to be a feature of neurosecretory cells, was first noted in these
particular cells in pulmonates by Flemming (1870, 1872). Other investigators
(Jobert, 1871; Huyguenin, 1872; Simroth, 1876, 1891; Retzius, 1892;
Beutler, 1901; Yung, 1911; Beck, 1912; Eckardt, 1914; Wille, 1915; Hoffmann, 1925; Bronn, 1928; Baecker, 1932; Demal, 1955; Tuzet, Sanchez, and
Pavans de Ceccatty, 1957) briefly referred to the existence of these tentacular
cells and made various suggestions as to their functional significance, but none
of them carried out any extensive examination of their cytoplasmic inclusions.
Experiments involving severance of the tentacles, and injection of extracts of
the distal portion of the optic tentacles into intact animals (Pelluet and Lane,
1961), indicated that in the slug Arion a hormonal substance is produced
by the tentacles that affects the cytodifferentiation of the ovotestis.
[Quart. J. micr. Sci., Vol. 105, pt. 1, pp. 35-47, 1964.]
36
Lane—Secretory cells in optic tentacles of Helix
The distal end of each optic tentacle of a typical stylommatophoran pulmonate, such as the snail Helix aspersa, contains a central ganglion around which
these apparently neurosecretory cells are arranged. Because of their distribution, I have called them the 'collar' cells (Lane, 1962). These cells, larger than
the other neurones within the ganglion, measure from 40 to 70/J, in length,
and are full of spheroidal inclusions that vary in diameter from less than 1 /x
to 2 or 3 /x. The collar cells are bipolar; one process extends to the base of the
epithelium that covers the tentacle, and the other merges into the ganglionic
mass.
A histochemical investigation carried out on the collar cells (Lane, 1962)
indicated that the two main visible cytoplasmic inclusions, the spheroidal
granules or a-bodies and the dispersed droplets or /3-bodies, both contain
lipid. In addition, peri-nuclear dictyosomes, up to 2 or 3 JJ, in length, are
present after prolonged osmication; the classical Golgi metal impregnation
techniques also produce smaller dictyosomes that are dispersed throughout
the perikaryon and lie between the a-bodies. These smaller dictyosomes were
interpreted at first as the osmiophil cortices of the dispersed lipid droplets
(Lane, 1962). However, the possibility remained that some of the smaller
dictyosomes might instead correspond to a Golgi lamellar-vacuolar field at
the ultrastructural level. Hence, an electron microscopical study was undertaken in order to determine whether any lamellar Golgi complexes were
present, and whether the peri-nuclear dictyosomes corresponded to any
recognized cytoplasmic component.
Light microscopical staining techniques for visualizing mitochondria gave
no interpretable results in the collar cells, since the a-bodies obscured the
ground cytoplasm to a large extent. Although it seemed improbable that
mitochondria were entirely absent from the cells, I wished to establish definitely their presence or absence, and in the case of their presence, to determine
the details of their fine structure, size, and distribution by electron microscopical examination.
I also wanted to investigate the ultrastructure of the spheroidal lipid inclusions in the tentacular collar cells of Helix, so that comparisons could be
made with the lipid globules of the cerebral neurones of the same species. In
addition, it seemed useful to compare the cytoplasmic inclusions of the collar
cells with the secretory inclusions of various other neurosecretory cells, to
ascertain in what ways the structure of the collar cells corresponds to that
considered typical of incretory neurones (as in Bern, Nishioka, and Hagadorn,
i9 62 )In the present study I have also carried out a brief investigation into the
fine structure of the neuronal processes in the tentacular ganglion, in order to
contrast their features with those of the neurosecretory axons and axon
terminations in other systems. I have also examined the lateral secretory cells
which line the dermo-muscular sheath investing the optic tentacles, to
elucidate the features of their ultrastructure.
Lane—Secretory cells in optic tentacles of Helix
37
Material and methods
The common garden snail, Helix aspersa Miiller, was used in this study.
The distal ends of the optic tentacles were dissected out and placed at once
in Palade's buffered osmium fixative (1952) at about 4 0 C for 50 min. The
tissue was dehydrated by treatment with an ascending series of alcohols,
followed by further dehydration with propylene oxide, and embedding in
araldite according to Luft's technique (1961). In some cases the material was
embedded in vestopal; when this was done, the tissue was treated with 0-5%
phosphotungstic acid for 1 h during dehydration.
Since the tissue was not a homogeneous one, sections were first made from
the hardened blocks with a sharp razor by hand, or with a sledge microtome.
These were mounted and examined by the light microscope, in order to find
the area of the block containing the cells to be studied. The blocks were then
pared down and sections cut on a Huxley ultramicrotome. Sections showing
silver or gold interference colours were mounted on formvar-filmed, carboncoated grids, or on uncoated grids. In some cases, but not all, the sections
were stained before examination, to increase the contrast; when staining,
Lawn's technique (i960) with potassium permanganate was used. The
sections were examined under an Akashi electron microscope (model TRS
50 El), operated at 50 kV.
From each block a few sections were cut at 0-25 n, after the silver or gold
sections had been cut; these were mounted on pieces of thin glass coverslips,
dried, and stained for \ min with a 1 % solution of toluidine blue / borax at
about 6o° C, as described by Meek (1963). These sections were mounted in
liquid paraffin under coverslips and examined by light microscopy in order
to determine the presence and localization of the particular cells being
investigated.
Results
The anatomical details of the optic tentacles of H. aspersa, the structure of
the secretory cells that are present in these tentacles, and the histochemical
reactions of the secretory cells have been described elsewhere (Lane, 1962).
Collar cells
The cytoplasm of the collar cells is difficult to study by light microscopy
because of the many spheroidal inclusions that fill the cell body; at the ultrastructural level, however, the cytoplasmic components can be distinguished
without difficulty, and inclusions not previously seen (Lane, 1962) can be
studied.
In addition to the spheroidal granules or 'a-bodies', which fill the bulk of
the cell body in most collar cells, there are also arrays of endoplasmic reticulum, mitochondria, Golgi complexes, vesicles of various types and sizes,
and 'jS-bodies'.
The a-bodies measure from 1 to 2^5 /A in diameter. They are of low electron
density and contain a number of electron-lucent vesicles, each about 60 m/x in
38
Lane—Secretory cells in optic tentacles of Helix
diameter (fig. 1, A, F). Circles or crescents are occasionally found within the
a-bodies (ec in fig. 1, F) ; these appear to be no more than the result of sections
cut through a projection or indentation in their surface. In some cases the
a-bodies have a crenated outline (fig. 1, c); in others, bulbous protuberances
are evident (pr in fig. i, B). Some of the a-bodies are linked one to another,
like beads (fig. 1, B); this phenomenon, and the way in which the endoplasmic
reticulum is sometimes arranged in relation to them, suggests that the cavity
of the a-bodies may be continuous with the intracisternal space of the endoplasmic reticulum. The intracisternal spaces are sometimes almost as large
as the smaller a-bodies, and they likewise contain non-electron-dense vesicles
(ic in fig. 1, c, F). However, the intracisternal spaces are always irregular, not
spheroidal, in shape; and if a living cell is ruptured, the a-bodies spill out
without any apparent connexion to one another (Lane, 19636).
The membranes of the endoplasmic reticulum, which are studded with
ribonucleoprotein granules, are continuous with the outer membrane of the
nuclear envelope (fig. 1, A). Cytoplasmic infoldings (|3-cytomembranes),
formed by invaginations of the plasma membrane, are present at the periphery
of the collar cells.
The mitochondria are rod-like or filamentous (fig. 1, A) and measure up to
11 jit in length and 0-33^1 in diameter. They are also present in the form of
circular profiles (fig. 1, A, B), which suggests that some of them may be spherical (although these may be cross-sections of filamentous forms). The cristae
of the mitochondria measure about 20 rap in thickness and often extend across
the whole width of a mitochondrion; between them, electron-dense granules,
20 m[x, or more in diameter, are sometimes found. The mitochondria are
scattered at random throughout the cytoplasm between the a-bodies, against
which they occasionally lie.
FIG. 1 (plate). All the micrographs shown in this figure are from the collar cells of the optic
tentacles of the snail, Helix aspersa.
A, section through the length of one cell which contains relatively few a-bodies (a). Note
the spherical andfilamentousmitochondria (m), the Golgi complexes (g), and the /3-bodies (|3).
Note also the nucleus (n) and those points (p) on its surface where its outer membrane links
up with the endoplasmic reticulum (er).
B, an area of peripheral cytoplasm showing arrays of endoplasmic reticulum (er), the membranes of which sometimes partially surround the mitochondria (TO). Note the Golgi complexes (g) and the a-bodies (<*) linked up in a moniliform way, by protuberances (pr) from
their surfaces.
c, cytoplasm containing an a-body (a) with a crenated outline, Golgi lamellae (g), j8-bodies
(j8) in different stages of development, containing varying numbers of electron-dense granules
(gr), and smaller circular profiles (c) which may give rise to the j3-bodies. ic, intracisternal
space of endoplasmic reticulum.
D, a j3-body (jB) containing parallel stacked lamellar membranes (/) as well as granular
inclusions.
E, the circular and kidney-shaped profiles (c) of developing jS-bodies. Note the membranebound electron-dense granules (eg).
F, cytoplasm with a-bodies (a), Golgi complexes (g), and a /3-body (/3) with electron-dense
granular inclusions (gr). Note the circular profile (ec) within one a-body, probably a transverse
section through an irregularity in its surface membrane, ic, intracisternal space of endoplasmic
reticulum.
FIG. I
N. J. LANE
Lane—Secretory cells in optic tentacles of Helix
39
Golgi complexes are found distributed evenly throughout the cell body
(g in fig. 1, A, B, c, F), in no greater abundance around the nucleus than elsewhere in the cytoplasm. In some cases two complexes lie close together, at
right angles to one another. They are composed of stacks of parallel, nongranular membranes, with vesicles at each edge and larger vacuoles lying to
one side (fig. 1, c). The lamellae are flat or slightly curved, and have an
average length of O-6JW., though in some cases they measure as much as 1*2/A
in length. The vesicles associated with them may be moderately electrondense.
Also found scattered through the cytoplasm are other vesicles that have a
rather intensely electron-dense core and an outer limiting membrane; these
measure about 75 m//. and are sometimes associated with the Golgi lamellae,
and sometimes not (fig. 1, E). They are often found close to electron-lucent
circular or kidney-shaped profiles that range in size from 0-16 to 0-38/J. (C in
figs. 1, c, E; 2, c, G), and that sometimes enclose a few smaller vesicles or
granules (fig. 2, c, G).
Larger circular or elliptical profiles, ranging from 0-5 to o-8 or 1 /x, are
distributed throughout the cytoplasm, three or more being often grouped near
each other. These, like the smaller profiles mentioned in the preceding paragraph, have a non-electron-dense matrix that contains a varying number of
electron-dense granules or vesicles, which range from 50 to 300 m/z. in
diameter and which sometimes have an outer limiting membrane (/? in fig. 1,
A, c, F). These electron-dense granules and vesicles in some cases are clumped
to one side within the profiles (fig. 1, c). Occasionally, parallel-stacked
lamellae are also found inside the circular or elliptical bodies (fig. 1, D). I shall
refer to these spheroidal or ellipsoidal objects, which resemble multivesicular
bodies, as /3-bodies.
Although under the light microscope the nuclei of the collar cells appear
circular or elliptical in section, under the electron microscope they have an
irregular outline. They contain a number of chromatin clumps, between
which diffuse granular chromatin is scattered (fig. 1, A).
Often the processes of 3 or more of the bipolar collar cells run alongside one
another in bundles towards the epithelial surface or the tentacular ganglion
(fig. 2, A). The epithelial surface, where one process of each collar cell terminates, displays a rather unusual structure: the epithelial cells have a number
of lacunae within and between them. In the tentacles of H. pomatia this has
been described by Schwalbach and Lickfeld (1962) as a basal labyrinth
system. An apparently extra-cellular fibrillar layer, about 3 (i thick, underlies
the free surface of the epithelial cells. The most superficial layer of all, just
above the fibrillar layer, is composed of microvilli (Lane, 1963a).
Those processes that run towards the ganglion appear, in one or two cases,
to run into it and to terminate in an area consisting of neuronal processes,
similar to those described below as typical of the tentacular ganglion.
Within the bundles of collar-cell processes, the plasma membranes can be
seen lying side by side, parallel to one another (fig. 2, A). In addition to
40
Lane—Secretory cells in optic tentacles of Helix
a-bodies and, sometimes, mitochondria, the processes contain non-electrondense vesicles from 45 to 145 m/x in diameter, thin filaments (which sometimes
look like thin tubules), and some electron-dense granules, 60 to 130 m/^ in
diameter, which often possess an outer limiting membrane. Occasionally
the processes also contain multivesicular bodies, of low electron density
{mv in fig. 2, E).
The bundles of collar cells in some cases have supporting or glial cells lying
around them. The peripheral cytoplasm of both the collar cells and the glial
cells is sometimes drawn into projections which protrude into the lacunae that
surround them; usually the collar and glial cells are separated from one
another at certain places by a portion of the lacunar space, and it is here,
within the cavity formed between the two cells, that the projections occur
(fig. 2, B).
Tentacular ganglion
Within the tentacular ganglion are found neuronal processes, which are
seen under the electron microscope in cross, oblique, or, less frequently,
longitudinal section. Most of these processes contain one or more circular
or rod-like mitochondria (m in figs. 2, D; 3, A, B, C) and a number of different
types of vesicles; they are sometimes protected by glial cells that lie around
and between them (fig. 2, D).
Four different types of vesicle are present in the processes, but not all types
are found in each. They are (1), electron-dense granules, about 10 to 20 m/A
in diameter, which are often aggregated in circular clumps (cv in fig. 3, c);
(2), intensely or moderately electron-dense granules with an outer limiting
membrane, up to about 140 m/x in diameter (eg in fig. 3, B, c); (3), nonelectron-dense vesicles measuring from 30 to 60 m/x in diameter (iv in fig. 3,
A, c); and (4), non-electron-dense vesicles, larger than (3) and from 80 to
120 m/x in diameter (Iv in fig. 3, A). The first type of granule is found in large
FIG. 2 (plate). The micrographs shown in this figure are from tissues in the optic tentacles
of the snail, H. aspersa.
A, section through the processes of collar cells with their plasma membranes (pm) lying
parallel to one another. Note the a-bodies (a), filaments (/) which in some cases look like
tubules, vesicles of low electron density (v), and electron-dense granules (eg).
B, glial cell (gc) and collar cell (cc) lying adjacent to one another, with a cavity (cv) formed
between them. Note the cytoplasmic projections from both the glial cell (gcp) and the collar
cell (ccp). I, lacunar space surrounding the cells; a, a-bodies.
C, cytoplasm with an a-body (a) and the circular profiles (c) of developing /S-bodies, with
vesicular and granular inclusions (gr).
D, transverse sections of neuronal processes (np) of the tentacular ganglion. Note the
mitochondria (m) and vesicles within the processes, and the glial cells (gl) running between
the processes; note also the capillary (ca) running through them.
E, neuronal processes from the tentacular ganglion. Note the multivesicular bodies (mv). m,
mitochondria; n, nucleus of an adjacent neurone.
F, section through a neurone lying on the periphery of the tentacular ganglion. Note its
nucleus (n), vesicular cytoplasmic components (v), and electron-dense, crenated, triglyceride
droplets (td). Some neuronal processes lie near it, including one which contains vesicles (dv)
that may be transverse sections through the continuous neurotubuli of a dendrite.
G, cytoplasm containing a-bodies (a) and circular or kidney-shaped profiles (c) that enclose
various inclusions (gr).
oc
Lane—Secretory cells in optic tentacles of Helix
41
numbers in nearly all the processes, the second in smaller numbers, and only
in certain of them. The third type is restricted to a few processes, while the
fourth occurs in a great many; they are alike in that they are usually found in
association with the first type, rather than with the second or each other; the
former, however, is found in profusion, while the latter only in single instances.
Thin filaments associated with the vesicles and granules can be observed in all
the processes (fig. 3, A, B, C).
Neurones measuring about IO/J, in length lie around the immediate periphery of the tentacular ganglion, as described in light microscopical preparations (Lane, 1962). They contain droplets that are electron-dense and
crenated, and therefore appear to be triglyceride (fig. 2, F). They sometimes
also contain other lipid droplets similar to those on the cerebral neurones of
Helix (Chou and Meek, 1958; Dalton, i960; Meek and Lane, 1963). I have
not observed in these neurones any electron-dense granules as small as those
in the neuronal processes of the ganglion.
Lateral tentacular cells
The optic tentacles of H. aspersa and other stylommatophoran gastropods
possess a number of secretory cells that lie along the inner edge of the dermomuscular sheath encasing each tentacle. In light microscopical preparations
these secretory cells have been divided into two categories, the 'lateral oval'
cells and the 'lateral processed' cells, which have been found to have different
histochemical properties (Lane, 1962). The lateral oval cells contain globules
which give a negative reaction to the periodic acid/Schiff (PAS) test for
polysaccharides, and are non-chromotropic; the globules of the lateral processed cells, however, are positive to the PAS test and exhibit metachromasy.
In spite of these histochemical differences, I have not been able to distinguish
between them under the electron microscope, so that the following description, as far as I have been able to ascertain, applies to both cell types. On the
other hand, the dermo-muscular sheath is lined mainly with lateral oval cells
close to the tips of the tentacles, and it is this area that I have chiefly studied.
The lateral cells contain a great number of electron-lucent globules that
measure from 1 to 3 ju. in diameter. These are circular or elliptical and sometimes contain a faintly granular substance, although usually they appear to
be empty (fig. 4, A, D, E, F). They often seem to be continuous one with
another (fig. 4, E).
In various parts of the cells, much smaller but similar globules are present,
often in close association with a Golgi complex (fig. 4, A), which appears to be
the site of their production. Because the globules fill most of the cell-body,
there is little endoplasmic reticulum, what there is has an extremely heavy
border of ribonucleoprotein granules along its component membranes (fig. 4,
A, D, F). A few scattered, circular, or filamentous mitochondria lie between
the globules (fig. 4, D).
Also dispersed between the globules, though infrequently and with no
specific orientation, are elliptical bodies, usually about 1 z to 17 JX in diameter;
42
Lane—Secretory cells in optic tentacles of Helix
they are distinguishable from the other globules by their content of electrondense inclusions, granular or rod-like in structure, and are limited by a single
bounding membrane, often difficult to resolve. In one case (fig. 4, E), a
lamellated circular inclusion was present, rather similar in structure to a
phospholipid droplet.
Discussion
An examination of the fine structure of the collar cells of the optic tentacles
reveals that they contain mitochondria which, because of their small size and
concentration between the a-bodies, could not be seen with the light microscope. On the other hand, light microscopical examinations did show osmiophil peri-nuclear dictyosomes (Lane, 1962), for which I can find no structural
basis under the electron microscope.
The smaller osmiophil and sudanophil dictyosomes found scattered through
the cytoplasm in light microscopical preparations (Lane, 1962) probably
correspond to the Golgi complexes and the jS-bodies seen in electron microscopical sections. Osmium-impregnated, light-microscopical preparations of
collar cells contain osmium deposited in the form of lumps or spheroids, in
addition to dictyosomes, which suggests that the /J-bodies, as well as the Golgi
lamellae, produce the 'dispersed lipid droplets' of light microscopy. The
dictyosomes were first considered to be duplex lipid globules with an osmiophil cortex and an osmiophobe core (Lane, 1962). It is improbable, however,
that they represent typical lipid droplets, for no inclusions containing concentrically arrayed lamellae and few containing parallel lamellae, characteristic
of phospholipid or mixed lipid globules (such as those found in the cerebral
neurones of Helix: Chou and Meek, 1958; Dalton, i960; Meek and Lane,
1963) were present in the cytoplasm of the collar cells at the ultrastructural
level. However, a lamellated body has been observed in a lateral secretory
cell, and Dalton (i960), studying the cytoplasm of the immature ova of the
snail, interpreted as transformed lipid droplets certain multivesicular bodies
to which the /J-bodies show some similarities.
An early stage in the formation of the ^3-bodies may be represented by the
electron-lucent, circular, or kidney-shaped profiles, o-16 to 0-38 /u. in diameter,
FIG. 3 (plate). All the micrographs shown in this figure are from the ganglia in the optic
tentacles of H. aspersa.
A, transverse and oblique sections of neuronal processes (tip). Note the mitochondria (?n),
vesicles of three sizes (v), (iv), (to). The intermediate-sized vesicles (iv) are clustered in certain
processes. Note also the filaments (/) in the axons and the neurotubuli (dt) of a dendrite.
B, transverse and oblique sections through neuronal processes. Note mitochondria (m),
filaments (/), and non-electron-dense vesicles of different size ranges (v, to); note also the large
numbers of membrane-bound electron-dense granules {eg), aggregated in certain axons.
c, transverse sections through neuronal processes (np). Note mitochondria (m), small
vesicles that may be aggregated in clumps (cv), and larger electron-lucent vesicles (Iv); one
process contains non-electron-dense vesicles (it)) that have a size intermediate between those
of the other two types of vesicle. Note also membrane-bound granules (eg) with an intensely
or moderately electron-dense core, as well asfilaments(/), which are often associated with the
vesicles.
FIG.
3
N. J. LANE
Lane—Secretory cells in optic tentacles of Helix
43
which are often found in association with the membrane-bound electrondense granules; with an increase in the size of these bodies, there seems to be
a corresponding increase in the number of granules and vesicles they contain
{as in fig. 1, c; 2, c, G). Gradual growth and transformation, by accumulation
of granules and vesicles, may therefore occur, leading to the ultimate production of a typical /3-body (such as in fig. 1, c). Groups of j8-bodies in different
stages of development are found in close proximity to one another within the
collar cells, which indicates either that maturation in different bodies occurs
at different rates, or that there is a continual production of new, immature
fi-bodies that develop at a constant rate.
The electron-dense inclusions that accumulate within the /3-bodies correspond in size range, and in some cases in structure, to the elementary neurosecretory granules described elsewhere in both vertebrate and invertebrate
neurosecretory systems (Knowles, i960; Bern, Nishioka, and Hagadorn,
1962). Hence it is possible that it is the /3-bodies that give the positive reaction
to staining for neurosecretory substances with alcian blue and chrome haematoxylin phloxine, found, in light microscopical preparations, in bodies scattered through the cytoplasm (Lane, 1962). Since the /J-bodies are not found
in the processes of the collar cells, while elementary granules are, it may be
that the j8-bodies store the granules before their release into the processes.
Multivesiculate bodies, of the same size range as the /3-bodies, and containing
accumulations of elementary neurosecretory granules, have been described
in the neurosecretory systems of other invertebrates (Knowles, 1962; Rosenbluth, 1963).
The j8-bodies resemble globules found in the neurones of some vertebrates,
in that they contain electron-dense granules and, at times, lamellar membranous profiles; in most cases, these vertebrate globules appear to be more
like lipid droplets than aggregations of neurosecretory material (Dalton, i960;
Murakami, 1962; Bern and Takasugi, 1962; von Harnack and Lederis, 1962;
Lederis, 1963; Afzelius and Fridberg, 1963; Fridberg, 1963). It has been
suggested, however, that in some secreting neurones, as in those of the goldfish preoptic nucleus (Palay, i960), a gradual transformation of multivesicular
FIG. 4 (plate). All the micrographs included in this figure are from the lateral cells lining
the sheath of the optic tentacles in H. aspersa.
A, cytoplasm containing electron-lucent globules (lg) of different sizes that are closely
associated with a Golgi complex (g), the lamellae of which appear to produce them; note also
the endoplasmic reticulum (er), studded with ribosomes.
B, cytoplasm containing an electron-dense inclusion (dg) lying amid the electron-lucent
globules (lg).
C, cytoplasm with electron-lucent globules (lg) and an electron-dense body (dg), perhaps a
lysosome.
D, cytoplasm containing electron-lucent globules (lg), mitochondria (m), and endoplasmic
reticulum (er) with a heavy border of ribosomes.
E, electron-lucent globules (lg) lying among endoplasmic reticulum (er) with an electrondense inclusion (dg) and a lamellar phospholipid droplet (pi). The dotted line indicates where
the globules appear to be continuous with one another.
F, cytoplasm with globules (lg) of different sizes, and endoplasmic reticulum (er) laden with
ribosomes.
44
Lane—Secretory cells in optic tentacles of Helix
bodies may occur. Palay believed that accumulation of electron-dense
material produced an electron-dense droplet visible under the light microscope. My suggestion concerning the development of the /3-bodies is a rather
similar one. In the droplets, Palay occasionally found elementary neurosecretory granules and profiles of packed flat membranes, like those described
in some j8-bodies, which he suggested showed similarities to the lysosomes of
other cell types.
It is noteworthy that the fine structure of the /3-bodies shows similarities
both to that of typical vertebrate lysosomes (such as those described by Essner
and Novikoff, 1962, and by de Duve, 1963), and to that of multivesicular
bodies, which are considered to be a type of lysosome (Novikoff, 1961). To
investigate further the possibility that the /3-bodies of the collar cells correspond to lysosomes, enzymatic tests are being undertaken to determine the
cytoplasmic site of acid phosphatase activity (the 'marker' or chief enzyme in
lysosomes) in the tentacular secretory cells.
Present in some of the collar-cell processes that run to the tip of the
tentacles are neurotubuli, typical of dendrites (Gray, 1959; Fridberg, 1963);
the processes may therefore be dendritic in nature. If this is so, the collar
cells may represent modified sensory neurones, and the processes running to
the ganglion would be axonal. The apparent connexion of these processes
with the tentacular ganglionic area suggests perhaps that some of the axons
in the ganglion arise from the collar cells.
Some of these axons contain membrane-bound, moderately or intensely
electron-dense granules (type 2), which correspond to elementary neurosecretory granules. It is possible that the particular axons that contain the
electron-dense granules originate from the collar cells since none of the
smaller neurones round the periphery of the ganglion have been seen to
possess elementary granules. No storage organ has been found, but bloodvessels run past the ganglion and coelomic sinuses surround it, so that secretory products could easily be extruded into the body fluids. In the sections
examined here, no region was found where the axons terminated on a specific
basement membrane adjacent to a blood-vessel, although processes have been
seen lying adjacent to a capillary (fig. 2, D).
In the ganglionic neuronal processes, the electron-lucent vesicles, 30 to
60 m/x in diameter (type 3), correspond to the 'synaptic' vesicles described in
axon terminals in other nervous systems (Dalton, i960; Holmes and Knowles,
i960; Fridberg, 1963; von Harnack and Lederis, 1962; de Robertis, 1962;
Hagadorn and others, 1963).
A process containing what appear to be vesicles, 22 mju. in diameter, is
shown lying near a peripheral neurone (fig. 2, F). Since the vesicles are
regularly arranged within the process, it is possible that what appear to be
vesicles are in reality cross-sections of the neurotubuli of dendrites; the typical
diameter of such a neurotubulus is said to be about 23 m/x (Gray, 1959). It
has been suggested that such cross-sections of continuous tubules within
dendrites are often mistaken for vesicles (Gray, 1959).
Lane—Secretory cells in optic tentacles of Helix
45
The vesicles of low electron-density, 80 to 120 m/M in diameter (type 4), are
of a size range comparable to some found in vertebrate neurohypophyseal
axonal endings, such as those described as 'light* or electron-lucent neurosecretory vesicles. (Kobayashi, Bern, Nishioka, and Hyodo, 1961; Heller and
Lederis, 1962). Some of the small electron-dense granules (type 1) probably
correspond to cross-sections of axonal neurofilaments, which Gray (1959)
defined as being 10 mjii in diameter.
In a discussion on the nature of neurosecretory systems, Bern, Nishioka,
and Hagadorn (1962) stated that the presence of typical elementary neurosecretory granules within a cell is not a necessary prerequisite for active
hormonal production; other particles, not the same as the elementary granules
and often larger than them, have been described in neurosecretory systems
and these may 'prove to be associated with hormonal entities' (Bern and
others, 1962). The cytological organization of the collar cells does not correspond to that of a typical neurosecretory cell, which contains a vast number
of separate elementary granules, obviously elaborated by vesiculation from the
Golgi lamellae (Bern and others, 1962). Certain facts, however, suggest that
the collar cells may be a form of neurosecretory cell. First, a hormonal subStance has been shown to be produced by the distal portion of the optic
tentacles of pulmonates (Pelluet and Lane, 1961), and no other cell-type in
that area has been found which could be the source of the hormone; secondly,
light microscopical investigations indicate that the collar cells merge into the
tentacular ganglion and may be modified neurones; thirdly, the j8-bodies (or
some other cytological component with a similar distribution and size) are
stained by the alcian blue and chrome haematoxylin phloxine techniques for
neurosecretion; and fourthly, the /3-bodies contain electron-dense vesicles,
some of which appear to correspond to the elementary neurosecretory
granules. The distribution of the lateral cells down the entire length of the
tentacles, and the fact that only the distal portion of the optic tentacles was
found to be hormonally active, suggests that the lateral cells cannot produce
the hormone affecting the ovotestis.
Upon first examining the lateral cells, I believed them to be peripheral
nerve-cells, as did Flemming (1872), because of their processed bipolar
structure and connexions with tentacular nerve-fibres. Their fine structure,
however, is remarkably similar to that of typical mucus-secreting cells, such
as the intestinal goblet cells (Palay, 1958; Florey, i960; Shearman and Muir,
i960) and the tracheal ciliated mucosa (Rhodin and Dalhamn, 1956) of the
rat. The size of the globules, the apparent continuity of the globules one with
another, their production by the Golgi lamellae, and the large numbers of
ribonucleoprotein granules on the membranes of the endoplasmic reticulum,
are all features which the lateral cells have in common with typical mucus
cells. However, the lateral oval cells are neither chromotropic nor positive to
the PAS test, as are the lateral processed cells and mucus goblet cells. Thus,
no definite conclusion can be reached.
The structure of the scattered electron-dense bodies in the lateral cells is
46
Lane—Secretory cells in optic tentacles of Helix
very similar to that of the lysosomes of some vertebrate cells. Also showing
structural similarities to lysosomes are the 'large opaque granules' described
by Rhodin and Dalhamn (1956) in rat tracheal mucus cells, and a queried
electron-dense body present in one of the goblet cells of the rat colon (Florey,
i960); Florey's body almost exactly resembles the electron-dense bodies of
the lateral cells (as in fig. 4, B, C). Rhodin and Dalhamn (1956) also described
a granule with concentrically arranged opaque membranes, similar to the
phospholipid lamellated inclusion of one lateral cell (fig. 4, E). These examples
are further indications of the similarity of the lateral cells to mucus-secreting
cells, and also suggest that the electron-dense bodies in the lateral cells, like
the /3-bodies of the collar cells, are in fact a form of lysosome.
In comparison with the lateral cells, the collar cells have smaller electronlucent globules (a-bodies) and a larger area of cytoplasm relative to them. In
contrast to the collar cells, the /3-bodies of the lateral cells sometimes contain
rod-like inclusions, but never flat lamellar membranes. There is, however,
a certain superficial similarity between the two cell-types. Clark (1956) has
described in annelids a situation where mucus cells seem to have evolved into
neurosecretory cells by increasing in duct length, sinking from the superficial
epidermal layers to the level of the nervous system, and being incorporated
into it. During this last stage of incorporation the cells develop the characteristics of neurones. Alternatively, it has been suggested (Scharrer and
Scharrer, 1937; Hanstrom; 1954) that in some neurosecretory systems, nervecells may evolve glandular characteristics. Either hypothesis would account
for some of the unusual features of the collar cells.
I am grateful to Dr. J. R. Baker, F.R.S., for his supervision and guidance
during the course of this work. I wish to thank Professor J. W. S. Pringle,
F.R.S., for accommodation in his Department. The Akashi electron microscope and Edwards vacuum evaporator used in this investigation were provided
by the Wellcome Trustees, and the Huxley ultramicrotome by the Royal
Society (grants to Dr. J. R. Baker). The financial assistance of the Canadian
Federation of University Women is gratefully acknowledged; this research
was carried out during the tenure of their Travelling Fellowship. My thanks
also are extended to the Pergamon Press Limited, for permission to use two
micrographs originally printed in Comparative neurochemistry.
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