<oologicalJournal
of
the Lznnean Socieg (1996), 116: 7 1-84. With 6 figures
Tardigrade biology. Edited by S. J . A4cInne.s and D. B. Norman
@
The cerebral ganglia of Milnesium tardigradum
Doy&re(Apochela, Tardigrada): Three
dimensional reconstruction and notes on their
ultrastructure
HOLGER WIEDERHOFT AND HARTMUT GREVEN*
Institut3r <oomorphologie und <ellbioloP;e der Universitat Diisseldo~0-40225 Dusseldod
Universitatsstr. 1, Germay
Differential interference contrast micrographs from stretched animals, serially sectioned semi-thin and
ultrathin sections revealed that the cerebral ganglia (supraoesophageal mass) of the eutardigrade
Milneslum turdigradum lie above the buccal tube and adjacent tissue like a saddle. It has an anterior
indentation which is penetrated by two muscles that arise from the cuticle of the forehead. The cerebral
ganglia consist of lateral outer lobes bearing an eye on each side, and two inner lobes which extend
caudally. Between the inner lobes a cone-like projection tapers into a nerve bundle. Each outer lobe is
joined with the first ventral ganglion. From the outer lobe near the eye the ganglion for a posterolateral
sensory field extends to the epidermis. Anterior to the supraoesophageal mass are three dorsal ganglia
for the upper three peribuccal papillae. Two additional ganglia attached to the cerebral mass supply the
lateral cephalic papillae. The cerebral ganglia are covered by a thin neural lamella. The pericarya which
surround the neuropil have large nuclei. Near the axons in the centre of the supraoesophagealmass the
cytoplasm is crowded with vesicles of different size and appearance. Some of them resemble synaptic
vesicles while others resemble dense core bodies. Structurally diKerent types of synapses and axons can
be distinguished within the neuropil.
01996 The Linnean Society ofLondon
ADDITIONAL KEY WORDS: -Eutardigrada
synapses.
~
supraoesophageal mass
-
neuropil
~
pericarya
~
CONTENTS
Introduction . . . . . . . . .
Material and methods . . . . . .
Light microscopy . . . . . .
Transmission electron microscopy
Abbreviations . . . . . . . . .
Results . . . . . . . . . . .
Anatomy and histology . . . .
Fine structure . . . . . . .
Discussion . . . . . . . . . .
Acknowledgements . . . . . . .
References . . . . . . . . . .
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*Correspondence to H. Greven
0024-4082/96/010071+14 $18.00/0
71
01996 The Linnean Society of London
72
H. IVIEDERHOFT AND H. GREVEN
INTRODUC7'ION
At the light microscopic level the basic organization and structure of the nervous
system of Tardigrada has been known since the last century (Doyi.re, 1840; Greeff,
1865; Plate, 1889; Lance, 1896; Basse, 1905; Thulin, 1928; Marcus, 1929;
Weglarska, 1975; Greven, 1980). Authors distinguished a cerebral ganglion
(supraoesophageal ganglion), with lateral lobes joined by connectives around the
anterior gut (circumbuccal ring, circumoesophageal connectives) to a suboesophageal ganglion and by circumpharyngeal connectives to the first ventral ganglion.
From the suboesophageal ganglion a double nerve cord extends posteriorly with four
ganglia along its length. In the anterior brain of Macrobiotu~sp. (Eutardigrada) a
cholinergic and peptidergic system was demonstrated by Raineri (1982, 1987).
At the ultrastructural level the ventral ganglia, some aspects of the cerebral
ganglion in 'l~urrobiotus-species(Greven & Kuhlmann, 1972; Weglarska, 1975), and
cephalic sense organs of Eu- and Heterotardigrada (Dewel & Clark, 1973;
Kristensen, 1981, 1982; Walz, 1978, 1979)have been described. Finally, Dewel et al.
(1993) provided a new description of the general organization of' the brain,
particularly of the eutardigrade Milnesium turdigradum. Following this survey the
central part of the nervous system is considered to consist of (1) a brain with a ring
neuropil and a posterior neuropil, both associated with ganglia, and (2) a chain of
four ventral ganglia. In addition, these authors gave details about the arrangement
of sensory receptors, ganglia and neuropils, and made suggestions concerning the
homology of nervous system components including those called deuto- and
protocerebrum in Heterotardigrada (Kristensen & Higgins, 1984 a,b). Very recently,
knowledge of the tardigrade brain has been deepened by a thorough study of the
brain in the heterotardigrade EchinZrcus viridissimus (Dewel & Dewel, 1996). This
report presents observations concerning the organization and fine structure of that
part of the brain of Milnesium tardgadum called the cerebral ganglion or
supraoesophageal ganglion by former authors. However, since the cerebral ganglion
is a composite of many ganglia (Dewel et al., 1993) the terms cerebral ganglia and
supraoesophagealmass are used here. We employed differential interference contrast
(DIC) microscopy of stretched animals and a computer-assisted 3-dimensional
reconstruction of serial semi-thin and ultrathin sections.
MATERI.4L .AND METHODS
Light microscopy
For light microscopy specimens of Milntsium turdgradurn, collected from moss on a
roof, were stretched maximally by removing oxygen (see Marcus, 1929) or by being
placed in a drop of 0.05 mol/l sodium azide for 1 to 2 h. Specimens were examined
in a drop of water with DIC microscopy.
For obtaining semi-thin and ultrathin sections stretched and unstretched
specimens were fixed either in 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer
(pH 7.2) or in 1 O/o or 2.5% glutaraldehyde plus 1 % osmium tetroxide in 0.1 mol/l
cacodylate buffer (pH 7.2) for 60-75min and postfixed in 2% osmium tetroxide in
the same buffer (60min). To facilitate penetration of the fixative, animals were
partially cut with a fine scalpel. Specimens were embedded either in styrene
CEREBRAL GANGLIA OF MILNESUM T m I G R A D L f i f
73
methacrylate (Kushida, 1961),LR-White (Newman & Hobot, 1987)or Spurr's epoxy
resin (Spurr, 1969). Series of 80 to 200 longitudinal or cross sections (0.5 pm in
thickness) from 1 1 different animals were stained in 19'0 Toluidineblue-Borax and
mounted in Eukitt. The method for the assemblage of the micrographs is described
by Wiederhoft & Greven (1989).
Transmission electron microscopy
Seven series of sections from specimens fixed with the simultaneous glutaraldehyde-osmium tetroxide fixation (each series consisted of 400 to 1200 single
sections) were placed on Pioloform/Formvar-coated 2 X 1 slotted copper grids,
stained with 4% aqueous uranylacetate followed by lead citrate and examined in a
Zeiss EM 9 at 60KV.
ABBREVIATIONS
als
cc
cg
cos
CP
e
il
anteriolateral sensory field
circumoesophageal
connectives
cerebral ganglia
circumoral sensory field
cephalic papilla
eye
inner lobe
m
ml
mu
n
mouth tube
median lobe
muscle
nucleus
nb
nl
nerve bundle
neural (basal) lamella
"P
01
Pg
Ph
Pk
neuropil
outer lobe
precerebral ganglion
Pharynx
ganglion of the posterolateral
sensory field
posterolateral sensory field
rostra1 papilla
stylet gland
stylet
PIS
Ip
S
st
RESULTS
Anatomy and histologu
The location, shape and dimensions of the cerebral ganglia of Milnesiurn tardigadum
are illustrated in Figures 1-4.. The cerebral ganglia lie saddle-like above the buccal
tube with its associated structures (stylet gland, stylets) and the anterior part of the
pharynx. The anterior portion of the 'saddle' has an indentation which is penetrated
by two muscles, which connect with the cuticle of the forehead. They extend
ventrally to the suboesophageal mass and attach dorsally to the posterior wall of the
pharynx (Fig. 1B.). An unpaired muscle joins the buccal tube dorsally. It originates
from the transition zone between buccal tube and pharynx and ends behind the
buccal lamellae (not illustrated). Caudally, the supraoesophageal mass shows two
roundish projections (inner lobes according to Marcus, 1929)and a median cone-like
projection (Fig. 2A,B), which tapers into a small nerve bundle. Paired small nerves
H. IVIEDERHOFT AND H. GREVEN
71
also arise from the inner face of the inner lobe (Fig. 1B). The circumoesophageal
connectives are visible ventrally at the anterior margin of the cerebral ganglia and
join the brain with the ‘suboesophageal ganglion’ (Fig. 1A). The buccal tube, stylets,
portions of the stylet glands (Fig. 2C) and four muscles that attach to the pharynx lie
within the circumbuccal ring formed by circumoesophageal connectives (Fig. 4B, C).
A
Figure 1. .\filniinpriurn /ard<qudun~
A. partial three-dimensional reconstruction of the supraoesophageal mass
and the ’snhoesophageal ganglion’. T h e inner lobes are hardly Lisible from this angle. Reconstruction was
asscinhlrd hy digitiziny each 0.5 pm section. B, diagram of the cerebral ganglia and associated structures,
frontal view. Nerve between outer lobe and first ventral ganglion (arrow). See Abbreviations for key.
CEREBRAL GANGLIA OF MILNESIUM TARDIGRADUM
75
In addition, there are two pairs of longitudinal muscles (dorsal)beneath each of the
inner lobes. The inner fibre of the muscle pairs extends from the dorsal aspect
beneath the cerebral ganglia to the anterior part of the buccal tube. Anterior to the
supraeosophaged mass, there are three precerebral ganglia (Fig. 2D). They innervate
Figure 2. Milmsium tardigradum (DIC). A-D, head in different planes to show the cerebral ganglia and
associated structures. E, first ventral ganglion. Tract from the outer lobe of the cerebral ganglia
(arrowhead).A-E, scale bar = 25 pm. See Abbreviations for key.
_.
/ t)
H. \VIEDERHOFT ,4ND H. GREVEN
the upper three of six peribuccal papillae. The middle dorsal ganglion attaches to the
two margins of the anterior indentation of the supraoesophageal mass, thus giving
Figurr 3. .\Ii/ntwm tnrdigrodurn. A. tangrntial section through the cerebral ganglia. Note peripheral
location of the nuclei (11) and neuropil (np). B, cross section. the outer lobe (01) with cyc (big arrow) and
rircurnoesophagd connectives icc!. .4.scale bar = 1 pm. B, scale bar = 5 pm, See Abbreviations for
key.
CEREBRAL GANGLIA OF MIIXESIUM TARLMGUUM
Figure 4.Milnesiwn turdigdzun. A, outer lobe (01) and circumoesophageal connectives (cc).B, same region,
note the ganglion (plg) of the posterolateral sensory field (pls). C, precerebral ganglion (pg) and parts of
the cerebral ganglia. Note the electron-lucent cell (arrowhead) which indicates the transition zone
between the outer lobe and the anteriolateral nerve bundle and the commissure of the precerebral
ganglion (pg). A,C, scale bar = 1 prn, B, scale bar = 5 pm. See Abbreviations for key.
77
78
H. WIEDERHOFT AND H. GREVEN
the impression of the presence of a hole. The two outer dorsolateral ganglia originate
from the transition zone of the cerebral ganglion and the circumoesophageal
connectives (Fig. 1B). At the same level, but more laterally, a voluminous nerve
bundle is present which branches beneath the cuticle forming an anterolateral
sensory field. The transition zone is characterized by a large electron-lucent cell in
the cerebral ganglia (Fig. 4C), which may also be seen in the light microscope. Two
additional ganglia, each of which joins to one ventrolateral cephalic papilla, arise
where the circumoesophageal connectivesjoin to the outer lobes. These outer lobes
form the lateral parts of the saddle. The posterior third of each lobe bears an eye
(Figs l B , ZC, 3B). The outer lobes are joined by two pairs of small ganglia. Each
elongated ganglion of the first pair arises in front of the eyes from the underside of
the outer lobe. They taper into paired vertical tracts which extend to the first ventral
ganglion anterior to two paired segmented nerves (Fig. 2E). Each ganglion of the
second pair arises with a small nerve at the upper side of the outer lobe, virtually at
the level of the eye, and is directed towards the epidermis to a posterolateral sensory
field (Figs 2B, C, 4B). At the site of origin this ganglion extends between a lateral
muscle and the epidermis. This muscle is the anterior branch of a bifurcate
transverse muscle (Thulin, 1928) - ‘dorsoventral muscles’ according to Marcus
(1929))‘sternodorsal muscles’ according to Plate (1 889) - which is attached to the
first leg and terminates anteriorly at the cuticle.
The semi-thin serial sections permitted the study of the distribution and number
of the large (2-3 pm in diameter) nuclei of the ganglion cells. Cell bodies with their
nuclei arranged in one to two layers cover the neuropil mainly dorsally of the
cerebral mass (Fig. 3A). The most common position in the circumoesophageal
connectives is at the front. In the supraoesophageal mass (without the outer lobes)
about 60 nuclei were counted in close proximity, only 1-2 pm from one another (Figs
3A, 5A). In the outer lobes the separation of pericarya and neuropil is not very
distinct, because pericarya with their nuclei (c. 30) are densely packed (Fig. 4A, B).
The ganglion for the posterolateral sensory field is about 15pm long and contains
usually 10 or more nuclei.
The precerebral ganglia which a diameter of about 8 pm consists of 8 to 9 cells.
In the lower ganglia with innervate the ventrolateral papillae large and translucent
cells are present, which are directed towards the circumoesophageal connectives.
Fine structure
Ultrastructural details of the cerebral ganglia are shown in Figures 3-6. The entire
cerebral ganglion is surrounded by a thin neural (basal) lamella (Figs 5A, 6A). Cell
bodies associated with the ganglia are large (about 5 pm in diameter), irregularly
shaped and have large nuclei and little cytoplasm. Nuclei (about 3 pm in diameter)
contain only a little heterochromatin and some electron-lucent holes of unknown
function. The nucleoli are located in close proximity to the inner membrane of the
nuclei. The cytoplasm contains numerous vesicles, RNA-particles, heterogenous
cytosomes, mitochondria, small amounts of rough endoplasmic reticulum and
dictyosomes (Figs 5A, 6A).
The neuropil consists of unsheathed nerve fibres, which contain fine filaments,
mitochrondria, a few microtubules and numerous vesicles. The vesicles - their
number increases from the origin of the axon to the centre of the neuropil - are
CEREBRAL GANGLIA OF MIWESIUM TMDIGRADUM
79
designated type 1,2,3 and 4 on the basis of their size and electron density (Figs 5, 6).
Type 1 (mean diameter 30nm) and type 2 (mean diameter 80nm) are electronlucent; type 3 (30-60 nm) and type 4 (50-70 nm) vary in electron density; a few have
dense centres and clear peripheries. Type 1 is concentrated on the presynaptic side
of the synaptic junctions and probably corresponds to synaptic vesicles (Fig. 6B,
inset). Type 2 are likely to be cholinergic vesicles (Fig. 6A). Types 3 and 4 (which are
perhaps only one type) resemble dense-core bodies which contain biogenic amines
(Figs 5A, 6A).
Nerve fibres which form the neuropil may be differentiated into four types (I-Iv)
Figure 5. Milnesium tardigradum. A, supraoesophageal mass. Neuropil with axons of type I, type I1 (with
vesicles of types 3 and 4), and synaptic contacts of type c. B, axon type IV.C, axon type 111. A X , scale
bar = 1 p.m. See Abbreviations for key.
H. \\'IEDERHOFT AND H. GREVEN
Figure ti. .\fih&m
kzrdigradutn. A. periphery and neuropil of the supraoesophageal mass. Axons (type 11),
vrsirles (t\prs 1 4 i . and s y a p s e s !qpe a,h). B, neuropil with different axons. Note synapses (a). Inset:
Syuapses hctwern similar axom (type b). .\,B. scale bar = 1 p.
CEREBRAL GANGLIA OF MIWESIUM TARDIGRALXIM
81
on the basis of their diameter and the amount of vesicles (Figs 5,6). Type I has a
diameter of about 250 nm, is electron-lucent and contains microtubules, mitochrondria and vesicles of Type 1 and 2 (Figs 5A, 6A). Type I1 has a more irregular outline
and is electron denser than type I. It contains vesicles of type 2, but the majority are
type 3 and 4 (Fig. 6A). Type I11 is fairly electron-lucent and flat; it is located largely
between the inner and outer lobes at the underside of the cerebral mass near the
neutral lamella (Fig. 5C). Type IV is relatively electron-dense and has a large
diameter; vesicles of all types are present and aggregations of vesicles occur near the
membranes of the axon (Fig. 5B).
Membranes of adjacent axons are connected by septate junctions. Synaptic
junctions are numerous. Three types may be distinguished, which are arbitrarily
designated as type a,b and c. Type a normally has dense bar-like material
incorporated into the membrane of its postsynaptic terminal. It is present when axon
I1 is adjacent to axon I (Fig. 6A). Type b is characterized by dense material
incorporated into the membrane of the presynaptic terminal between two axons of
type I; usually dense material of various dimensions is located also on the membrane
of its postsynaptic terminal. Both sides have many vesicles of type 1 and 2 (Fig. 6B,
inset). Type c is present when three nerve fibres of type I share a common
presynaptic terminal and dense material can be observed in the zones of contact
(dyades; Fig. 5A).
Precerebral ganglia are recognizable by a more heterogeneous structure. Many
cells are fairly electron-lucent. They are located preferentially at the side of the
ganglion which faces the cerebral ganglion. Other cells are more electron-dense.
Clear cells contain vesicles of type 2,3 and 4, mitochondria and rough endoplasmic
reticulum. Dark cells are similar to the cell bodies of the cerebral ganglia.
DISCUSSION
From our results obtained by DIC microscopy and three dimensional reconstruction of semi-thin sections, it becomes evident that the basic plan of the brain of M.
tardigradum corresponds to the summarizing drawings of Marcus (1929). However,
knowledge of brain ultrastructure requires not only a modified terminology, but also
a reinterpretation, as undertaken by Dewel et al. (1 993, personal communication)and
particularly by Dewel & Dewel (1996). In the first paper the authors discuss possible
homologies of the different regions of the brain in different tardigrade taxa and some
ultrastructural findings, in the second they suggest that the brain of tardigrades is
derived from the nervous elements of four segments and speculate on the evolution
of the arthropod head. With respect to the various ganglia associated with the
neuropil ring and the posterior neuropil (see Introduction) we will briefly discuss here
the outer lobes that correspond to the ganglia of anterior and posterior sensory fields,
the precerebral ganglia and some ultrastructural features.
In the head of M. tardkradum we identified a circumoral (cos), and anteriolateral
(als, including cephalic papillae),a posterolateral (pl), and a suboral (sos) sensory field,
and the pharyngeal organ (unpublished). They correspond to the sensory organs
(listed in the same order) under the circumoral cuticle, the oral papillae, the cephalic
papillae (anterior sensory plaque), the posterior sensory plaque, and the sensory
organs under the mouth ring as well as in pockets dorsal and ventral to the stylets
mentioned by Dewel et al. (1993) for the same species. Four of these sensory fields
w2
H. \tIEDERHOFT AND H. GREVEN
(COS, als, sos, pharyngeal organ) have previously been described for Macrobiotus
hufelandi by W’alz (1978). A ganglion between the outer lobes and the posterolateral
sensory field was not explicitly mentioned by Marcus (1929), Weglarska (1975) and
by Walz (1978, 1979). However, Weglarska (1975: 452) wrote: “Three lobes
(Marcus’ inner lobes) are distinctly visible. These lobes make a close whole and
undoubtedly form the main part of the brain. The outer lobes are connected to them
loosely. Their appearance differs from the scheme shown by Marcus (1929). Thin
out<growthsmade mainly of nerve filaments join them with the brain. The nuclei of
these lobes are shifted to the distal bulb-shaped extensions . ..”. Weglarska’s (1975)
‘outer lobes’, ‘loosely connected’ with the main part of the brain resemble the
ganqlion for the posterolateral sensory field (see also Dewel et al. 1993, who
homologize these lobes to the posterior portion of the outer lobes). The obvious
differences in the shape of the brain call for more detailed comparative studies.
Marcus (1929; see his Fig. 28a) refers to observations of Basse (1905) and Thulin
(1928) and he and Walz (1978, 1979) described three nerve fibres extending from the
anterior margin of the cerebral ganglia to the anteriolateral region of the head of hf.
hufelandi. They have not been referred to in M. tardzgadum. These results show that
in this species the anterolateral head region is supplied with nerve bundles on both
sides, which branch under the cuticle forming the anterolateral sensory field.
Nerves extending from the inner lobes in h%hujilandi (Basse 1905) also occur in M.
tardigradum. Marcus (1929), who was not able to identifjr these nerves, expresses
resemations about their possible presence. He cited Basse (1905), who believed that
these nerves innemate the stylets. It is suggested that they may innervate the two
four dorsal muscles
outermost of the dorsal trunk muscles. The two innermost
extending through the entire body are typical for Eutardigrada - are supported by
the nerve fibres of the median projection by means of a ‘triangular’ neural mass.
Precerebral ganglia named oral papillae ganglia (Dewel et al., 1993) or cone
ganglia Dewel & Dewel (1996) innemate the six peribuccal papillae. In the
hetcrotardigrade Echinisccw ciridissimus the ventral cone ganglia appear to represent
the suboesophageal ganglia. In this study we directed our attention mainly to the
‘supraoesophageal ganglion’ semu Marcus and the three upper precerebral ganglia.
However, some SEM preparations and 3-dimensional reconstructions of serial
sections indicate a similar arrangement in hl.tardeadurn.
The number of pericava of h.l. tmdkradum (estimated by counting the nuclei) was
about 60in in the inner and median lobes and about 30 per outer lobe. Adding 10
nuclei per precerebral ganglion and the 15 nuclei per ganglion for the posterolateral
sensoq- field there were a total of about 180 nuclei. Marcus (1 929) estimated for the
supra- and suboesophageal ganglion and the circumbuccal ring a number of 300 to
400 cells in Macrobiotus.
Occurrence and position of glial cells were not considered in our study and await
further studies. In a previous study on Macrobiotus hufelandi we recognize these cells by
their high numbers of ribosomes (Greven & Kuhlmann, 1972; see also Dewel et al.,
1993). Identification of glial elements in the central part of the brain in Milnesium
tardigradum based only on this criterion is dubious and should be sustained by other
tnethods (e.g. immuriocytochemistry) in the future.
The synaptic contacts resemble those of other invertebrates. For example, in
AVlicrvstomumlineare (“urbellaria) Reutter (198 1) described several types of synapses: (1)
a single synapse with presynaptic densities beneath a thickened presynaptic
membrane and a dense postsynaptic membrane; (2) a shared synapse which has a
~
CEREBRAL GANGLIA OF MILNESIUM TARDIGRADUM
83
presynaptic terminal contacting two postsynaptic elements; (3) an ‘en passant’
synapse characterized by parallel nerve fibres having clusters of vesicles against the
inner side of one of the limiting membranes; (4) a synapse without thickening on its
pre- and postsynaptic membranes. All of these synapses show a mosaic of electronlucent and electron-dense cored vesicles on their presynaptic sides (see also Greven
& Kuhlmann 1972).
Axons of the pericarya form the neuropil of the cerebral ganglia and contain
numerous vesicles. These vesicles, designated here arbitrarily as type 1 to 4,
correspond partly to synaptic vesicles (type 1). Type 3 and mainly 4 resemble dense
core vesicles which contain biogenic amines and/or peptidergic inclusions. Both are
localized in structurally similar vesicles in other invertebrates (Gersch & Richter,
1981).Axons with larger ‘empty’ vesicles (type 2) may correspond to the cholinergic
system. Raineri (1982, 1987)found acetylocholinesteraseactivity in the brain and the
ventral ganglia as well as evidence for peptidergic neurons in the nervous system of
Macrobiotus sp. Unfortunately her types of neurons designated as ‘pigmented’,
‘basophilic’, ‘clear’ and ‘small’ cannot be compared with that of M. tardigradum. In
addition, the relationship of the various neurons to the brain remained unclear. It is
difficult to link the diagrammatic representations of the brain in her paper with that
of the Eutardigrada. Cytochemical investigations, now in progress, will allow a better
differentiation and localization of the various neurons observed.
ACKNOWLEDGEMENTS
We should like to thank Dr U.J. Santore for critically reading the manuscript and
his suggestions for linguistic improvements and Drs R. A. and W. C. Dewel for
constructive critique.
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