1 NC
INTRODUCTION
The neural gland complex of ascidians is located in the
intersiphonal region between the branchial basket and the
body wall. This complex consists of a lobed gland, a ciliated
tubercle-funnel, and a canal connecting the two. The tubercle
is anterior to the gland and opens into the pharynx just
above the junction of the peripharyngeal grooves (figure 1).
The gland itself lies next to or near the ganglion, and a
tube called the dorsal strand runs posteriorly from the
gland and possibly to the gonads. Although the neural gland
has been observed since the late nineteenth century and has
been thoroughly reviewed by Ivan Goodbody (1974), its function
remains unknown. Many studies approach this urochordate
organ as a possible precursor or homolog to one of the
vertebrate gland systems, but essentially no evidence for any
such homology has been generated. Without disregarding
previous speculations, the present study is based on direct
observations of the ascidian neural gland complex in live
dissections and histological preparations. Some preliminary
histochemical and physiological tests provide suggestive
leads for further investigation of the nature of this gland.
MATERIALS AND METHODS
Ascidia ceratodes (Order Enterogona) was used for living
observations. Specimens were collected from dock floats in
the Monterey Marina during April and May and were kept in a
2 NC
running sea table at about 15° C.
The neural gland complex of Ascidia ceratodes lies ventral
to the ganglion (figure 2), hence the animal must be dissected
to get direct access to the gland. Dissections were per¬
formed by removing the animal from its tunic and cutting the
body wall lengthwise from the oral siphon down the left side
of the endostyle. Care was taken not to injure the heart or
other organs. The opened animal was then pinned with three
stainless steel minuten pins to a petri dish half-filled with
Sylgard (Dow-Corning), a soft, clear plastic. The dissections
could thus be viewed with illumination from all angles under
both dissecting and compounds microscopes.
Most histological examinations were performed on Styela
montereyensis (Order Pleurogona). In this species the gland
lies dorsal to the ganglion in a potentially convenient
location for external operations. Because the animal
chronically contracted in response to cutting the tunic,
however, attempts to approach the gland by shaving away the
tunic between the siphons were unsuccessful.
Several techniques were employed to observe the nature
of communication between the gland complex and the rest of
the animal. The dorsal tubercle provided the first site of
study. Currents around this ciliated tubercle-funnel were
traced by introducing a solution of graphite particles
suspended in phytoplankton culture near the tubercle of a
dissected preparation.
In order to test for possible exchange of material
3 NC
between gland and pharynx, aluminum foil crimps and silk
thread ties were used to ligate the canal. Jabbing a thin,
pointed piece of foil under the canal and folding the sides
up around the canal proved to be the most effective method
of ligation. The tentacles were trimmed and the pharynx cut
away as much as possible without disrupting either gland or
ganglion. Any gross changes in gland appearance were noted
until the animal died. Hearbeat and contraction of the
oral siphon were used as positive indications of viability.
Unligated dissections were observed concurrently as controls.
To test for movement of material into the gland via the
dorsal tubercle and canal, dissections with ligated and
unligated canals were soaked in either Neutral Red or
fluorescein solutions. Saturated solutions of these dyes
were prepared with filtered sea water and constant stirring
for 15-20 minutes with subsequent re-filtering. Dissections
were soaked in these dyes at 5-10° C in the dark overnight
and then rinsed in fresh running sea water for at least two
hours. Glands of the ligated and unligated specimens were
then compared.
To test for movement of material out of the gland via
the canal and tubercle, dissections were injected with
solutions of methylene blue, fluorescein, or Neutral Red
prepared as before. The canals were then ligated. Unligated,
injected preparations were used as controls. Injections were
administered using a micropipette pulled on a David Kopf
vertical pulling machine. This pipette was then attached
4 NC
to a hypodermic needle with a hot glue gun (Sears, Roebuck Inc).
The fine tip of the pipette was inserted down the canal via
the lower lip of the ciliated funnel and pressure was
applied manually with a bulb until stain was seen to enter
the gland.
In order to avoid artifacts introduced by the process of
dissection, experiments were also performed on intact animals.
Ten Ascidia ceratodes were soaked overnight in one of four
dye-sea water solutions: methylene blue, Neutral Red, methylene
blue plus Neutral Red, or fluorescein. The specimens were
then rinsed for several hours in fresh running sea water and
dissected. Glands were monitored until the preparations
died.
Preliminary enzyme assays were conducted on neural gland
extract in order to test for digestive activity in the gland.
Drops of the extract, prepared by grinding ten freshly
isolated glands in 1 ml. of sea water, were placed on the
emulsion side of exposed black-and-white photographic film
and on tiny pieces of semolina macaroni noodles (Golden Grain).
Drops of 0.1%, 0.01%, and 0.001% protease in sea water were
placed on the film and noodle substrates as controls. These
preparations were kept in a moisture chamber and observed
every hour for a few hours. They were then left in the
chambers overnight
Both A. ceratodes and Styela montereyensis were used for
histological studies. Rectangular pieces of body wall
including gland, tubercle, ganglion, and attached pharynx
5 NC
were fixed and embedded according to procedures outlined in
the data sheet of the JB-4 plastic embedding kit (Polysciences,
Inc. Warrington, PA). A specimen from Ascidia and one from
Styela were cut in 2-4 micron transverse sections and another
tyela specimen was cut in 2-4 micron longitudinal sections.
An average of fifteen slices were expanded in ammoniated water
on each microscope slide, after which the water was evaporated
off over a Bunsen burner. Slides with sections thus mounted
were then stained by the Lee's methylene-blue-basic fuchsin
method also outlined in the JB-4 embedding kit data sheet.
Giving stained slides a subsequent rinse in 95% ethanol for
roughly half a minute followed by a rinse in distilled water
proved effective for removing excess stain from the embedding
plastic. Balsam was used to mount the cover slips because
it has an optical density close to that of JB-4 plastic.
Slides were observed and photographed under a Zeiss compound
microscope.
Extract made from 15 glands of A. ceratodes ground in
I ml of sea water was applied to a smooth muscle preparation
of Ciona intestinalis used by Gabrielle Nevitt in her smooth
muscle studies of 1981. Twitch amplitude of the muscle strip
in 60mM calcium solution was first measured. Extract was next
applied to the preparation and the change in twitch ampli-
tude was determined. The gland extract was then washed out
with the high calcium sea water and full recovery was
obtained. The experiment was repeated using filtered sea
water as a control
6 NC
In order to test for the presence of biogenic monoamines
in the neural gland or surrounding tissues, specimens of A.
geratodes similar to those used in the histological studies
were treated with glyoxylic acid according to the technique
described by de la Torre and Surgeon (1976). Treated
specimens were cut on the cryostat into 10 micron serial
sections and mounted on microscope slides. These slides
were then examined under the UV fluorescence microscope with
various filters.
RESULTS
Observations on currents around the dorsal tubercle
Currents around the dorsal tubercle of Ascidia ceratodes
made a vortex-like pattern as shown in figure 3A. Particles
drawn towards the tubercle were thrown away from or swirled
back onto the tubercle. These graphite particles stuck to
mucous covering the tubercle and the graphite-laden mucous
formed a strand that moved towards the dorsal lamina,
ultimately joining the mucous strands of the peripharyngeal
grooves (figure 3B).
Observations on soaked and injected preparations
Of the five dissections soaked in Neutral Red dye, three
were ligated and two were unligated. All five showed obvious
staining in the pharynx, tubercle, and tentacles, but very
little, if any, in the gland. In a similar experiment using
fluorescein dye, ligated and unligated specimens were
examined in a dark room under a hand-held UV light. Although
7 NC
the ganglion, tubercle, pharynx, and blood tissues all glowed.
those of the neural gland did not. This would seem to
indicate that the gland had not sequestered fluorescein from
the surrounding water in either preparation, although the
bright glow of the ganglion may have overshone any glow
emanating from the gland.
Injection experiments gave erratic results. Sometimes
the dye went into the gland, sometimes into the dorsal vessel.
Once the dye was in the gland it usually stayed there, although
fluorescein was observed to fade, making detection difficult.
While retention of injected methylene blue could be caused by
specific staining of neural gland cells, retention of
fluorescein cannot be so easily explained. Either fluorescein
is actively taken up by the gland or else diffusional communi-
cation from the site of injection to the tubercle is limited.
Of the three dyes tried, fluorescein was the least retained.
These experiments are thus inconclusive but do suggest a
possibly fruitful, albeit technically difficult, avenue of
approach.
Whole soaked animals gave interesting results. Like
soaked injections, animals soaked in fluorescein glowed in
the ganglion, tubercle, pharynx, and blood when viewed under
UV light, but did not glow significantly in the gland.
Animals soaked in a mixture of Neutral Red and methylene blue
showed red staining of the pharynx, blood, and tubercle, but
the appearance of the gland varied. Some had glands full of
blue and red stained cells,yothers had glands virtually
(figure 1)
8 NC
unstained except for a few blood cells stained red, and still
others had glandsof intermediate appearance. One specimen
of particular interest had a fairly empty gland and a clot
of stained cells in the canal just below the tubercle.
Whole animals soaked in methylene blue had glands that
distinctly concentrated the blue color more than any other
organ or tissue, although the tunic stained dark blue. Once
again, different degrees of staining were observed. For
example, one gland was largely empty of stained cells and had
clear, bubble-like lobes rimmed with some stained cells, while
a few other glands were very compact and stained dark blue.
In one preparation a clump of blue cells was observed to
loosen from the gland, spiral up the canal, and go out the
ciliated funnel-tubercle. The clump then joined the mucous
strand on the tubercle and moved as part of the strand towards
the dorsal lamina. These discharged cells were very similar
in appearance to stained cells in the gland itself when the
two cell samples were examined under the compound microscope.
In summary, it would seem that fluorescein does not
get trapped in the lumen of the gland when applied externally,
but that Neutral Red and methylene blue can gain access to
the gland cells via some route and stain them. Unspecific
staining by Neutral Red in vital preparations make it a
poor choice for the appointed task, but methylene blue seems
to work well for semi-specific staining of the neural gland.
Observations on digestive activity of neural gland extract
Neural gland extract did not significantly digest film
emulsion or semolina noodles, even when left in a moisture
9 NC
chamber overnight at room temperature. All three concentrations
of protease digested both film emulsion and semolina noodles.
Observations of histological preparations
Many interesting features of the ganglions and neural
gland complexes of S. montereyensis and A. ceratodes were
observed in the 2-4 micron sections of the two species.
Longitudinal sections of Styela and transverse cuts of both
Styela and Ascidia were examined at 100X and 400X dry
magnification and 1000X oil-immersion magnification.
Although connective tissue separated gland and ganglion
over most of the gland-ganglion interface, sites of contact
where gland cells seemed to merge with those of the ganglion
and vice versa were evident in several places. One of these
sites was photographed through a Zeiss light microscope at
the three magnificationspreviously mentioned and is shown
in figures 4A through 4E. The sections depicted are 2-1
micron longitudinal slices through the neural gland and
ganglion of Styela montereyensis, and the entire series
spans a thickness of 20-30 microns.
Figure 4A shows part of a section oriented with the
dorsal surface at the top of the photograph. The gland
appears as a mottled triangular projection sandwiched between
the body wall above and the ganglion below. Part of the
ciliated funnel-tubercle can be seen on the ventral surface
of the ganglion. The area enclosed in the white box includes
a ganglion-gland cellular bridge and is viewed at higher
magnification in figures 4B through 4E.
10 NC
Figures 4B-4E are photos of four pseudo-serial longitudinal
sections through the gland-ganglion bridge in figure 4A at
higher (400X) magnification. The section shown in JE is
reversed, an artifact of its placement on the microscope
slide. Once again, ganglion lies below the dark, triangular
projection of gland tissue, and cells of the beginning of a
nerve can be seen at the top
of the photo. A connective
tissue layer separating the gland and ganglion can be seen
to the right of the cellular bridge, but this layer becomes
obscure and disappears where the gland and ganglion cells
merge.
Also of interest are the cell types in the gland and
ganglion. Both bear large, dark inclusions, and the
ganglion cells often seem to be empty. Gland cells in these
sections are not well delineated except for what appear to be
nuclei (the dark inclusions), however the cells are distinct
from those of the surrounding tissues, namely muscle and
ganglion. In regions where ganglion and gland cells
make direct contact, this distinction is often lost.
Observations on smooth muscle relaxant properties of gland
As outlined in the methods, extract of neural gland of
Ascidia ceratodes was applied to smooth muscle preparations
of Ciona intestinalis and changes in twitch force in response
to brief electrical shock were noted. The top panel of
figure 5 shows that gland extract seems to have caused the
contractions of the smooth muscle preparation to rapidly
decrease and stabilize at half the force of the control
contractions in high calcium solution. After reversing
11 NC
the gland extract effect, application of filtered sea water
as a control did not have a significant effect. (figure 5,
bottom panel). Contractions were transiently reduced and
stabilized at 90% of the force in high calcium. Neural gland
extract thus appears to have a reversible smooth muscle
relaxant effect in this preliminary experiment, and further
tests are desireable.
Observations on histofluorescence tests
Using the glyoxylic acid fluorescence technique of
de la Torre and Surgeon (1976), 10 micron longitudinal sections
of the neural gland region were examined under the UV
fluorescence microscope. The inner periphery of the ganglion
was seen to glow a greenish yellow when excited by light of
450 nm wavelength, but gland tissue was not observed to
fluoresce. Resolution in this preparation was not fine
enough to determine if gland cells in the gland-ganglion
bridge regions fluoresced. Cells in the interior of the
gland did not glow, although blood cells, probably vanado¬
cytes, fluoresced bright yellow.
DISCUSSION
Although none of the above testsconclusively demonstrate
the function of the ascidian neural gland complex, many of
the results provide useful starting points for further investi-
gations. The study also introduces some innovative techniques
for working with this elusive little organ.
12 NC
Vortex currents around the tubercle and the occurrence
of cells exiting the gland via the canal are supportive
evidence for earlier claims that material moves from the
gland and into the pharynx. Although cells leaving the gland
were seen to join the mucous food rope system, no digestive
enzymes were detected by crude assays performed on gland
extracts. It would appear that the gland does not function
as a digestive organ.
Histological observations made in this study indicate
that there are several sites of cellular connection, even
fusion, between the tunicate ganglion and neural gland of
two solitary ascidians, namely Styela montereyensis and
Ascidia ceratodes. Although resolution under the light
microscope was not fine enough to detect clear axonal
connections between the two organs, such intimate cellular
contact implies some form of communication. It is possible
that gland cells release substances to other tissues or organs,
for instance the ganglion, and are then discharged out the
canal and into the food rope, where they are eventually ingested.
The stained granular bodies seen in gland cells of animals
soaked in methylene blue may participate in such a secretory
process.
The relaxant effect of neural gland extract on tunicate
smooth muscle provides a clue as to the nature of the gland
cell contents. For instance, it is known that some monoamines
(Prosser 1974
have a relaxant effect on smooth muscle.A The presence of
biogenic monoamines in the neural gland complex or ganglion
123 NC
coupled with the evidence from histological peparations of
gland-ganglion cellular bridges, would suggest that the two
organs exchange substances related to neurotransmitters.
Results of the histofluorescence test partially support
such
speculations in that fluorescence, implying a corresponding
presence of biogenic monoamines, was observed around the
periphery of the ganglion. The test did not demonstrate the
presence of any such substances in the negral gland, however,
and repetitions of this experiment by experienced fluorescence
histochemists are desireable.
Continuation of the histofluorescence studies on the
neural gland complex and elaboration of the smooth muscle
experiments should be coupled with electron microscopy
of the gland-ganglion interface. Such studies would provide
valuable information on the chemical and structural nature
of this region. Depending on the results; this information
could then be used to determine the function of the tunicate
neural gland.
AKNOWLEDGEMENT
Many glandfuls of thanks go to Prof. William Ph.* Gilly
for his generous assistance, as well as to Prof. Donald P. Abbott
for his dedication and lessons in noodle enzymology. I would
especially like to thank Mr. Keith Kohatsu, the TWTA, for histological
heroism and general incredible output, and Chris Patton for yet
more histology instruction. Finally, I would like to thank my
classmates of the Bio 175H class 1981 and all others at Hopkins
who helped make this an outrageously enjoyable quarter
as in Pheromone
14 NC
LITERATURE CITED
Goodbody, I. (1974). The physiology of ascidians. Advances in
Marine Biology 12: 107-128.
Polysciences, Inc. (1976). JB-4 embedding kit, data sheet 123.
Paul Valley industrial Park, Warrington, PA. 18976.
Prosser, C.L. (1974). Smooth muscle. Annual Review of Physiology
36: 503-537.
de la Torre, J.C., Surgeon, J.W. (1976). A methodological approach to
rapid and sensitive monoamine histofluorescence using a modified
glyoxylic acid technique: SPG method. Histochemistry 19: 81-93.
15 NC
FIGURE LEGEND
Figure 1:
Ventral view of neural gland
complex in dissected
Ascidia ceratodes.
grooves (PP), dorsal
Peripharyngea
tubercle/ciliated funnel
canal (C), neural gland (NG),
(DT/CF).
ganglion (GG), and tentacles (T).
parkness due to staining with
methylene blue and Neutral Red.
Figure 2:
Drawing of longitudinal paraffin section through neural
gland complex and ganglion of A. ceratodes.
Figure 3:
A) Water currents around dorsal tubercle of dissected
A. ceratodes.
B) Mucous with graphite strands moving from tubercle and
joining mucous strands from peripharyngeal grooves.
Figure 1
A) Longitudinal section of Styela montereyensis at 100X
magnification showing neural gland (NG), ganglion (GG), and
part of the dorsal tubercle/ciliated funnel (DT/CF
Region in box shows gland-ganglion cellular bridge (CB).
B-E) Longitudinal sections of S. montereyensis at 400X
magnification- expansion of gland-ganglion bridge shown in
figure 4A. Figure HE is reversed.
Figure 5:
Polygraph record of relaxant effect of neural gland
extract (A. ceratodes) on smooth muscle (Ciona intestinalis).
Figure
16 NC
DORSAL
ganglion.
Ascidia ceratodes
longitudinal section
ANTERIOR
ciliated funnel
canal
VENTRAL
-neural gland

POSTERIOR
jure

17 NC
a


e
5
18 NC
dorsal tubercle
currents
-peripharyngeal groove

—to dorsal lamina
dorsal tubercle
mucous strand from tubercle
.


mucous strand
in peripharyngeal groove
clump of mucous
going to dorsal lamina
Figure 3
Figure 4
at/ef
19 NC
Figure 4
30 microns
30 microns
20 NC
Figure 4
30
microns
micro
21 NC
high Ca
6OmM

A
bigh Ca
6OmM
Extract of Ascidia ceratodes neural gland on
smooth muscle of Ciona intestinalis


neural gland extract

filtered sea water
Figure 5