Abstract
Cells isolated from optic lobe neural tissue from embryos of the
squid, Loligo opalesceng develop processes in culture. Growth of
neurites depends on pH of the culture medium, nature of the
substrate, and presence of medium additives (Fetal Bovine Serum,
squid blood, and hemolymph). The optimal conditions identified were
PH 7.7, poly-L-lysine substrate, and bovine serum. Both whole squid
blood and hemolymph depressed neurite outgrowth.
Introduction
In squid the giant axon controlling escape jetting is formed by
fusion of small axonal processes from hundreds of cell bodies in the
giant fiber lobe of the stellate ganglion (Young, 1939). Unlike the
giant axons to which they give rise, these cell bodies do not show any
sodium currents when studied with voltage clamp techiques in situ
or after enzymatic isolation. Sodium currents appear after several
days if the isolated cell bodies are grown in a cell culture and
appearance of Na channels depends on mRNA synthesis and
microtubules (Brismar and Gilly, 1987). However, very little is
known in regards to how these channels are targeted for transport
from the site of synthesis in the neuronal cell body to their final
destination in the giant axons. How such sorting occurs and what are
the regulatory factors in channel trafficking are important questions
to answer in trying to extend our knowledge of long-term nervous
system function. These questions and others, such as what directs
axonal growth towards the correct target, what mediates cell-cell
recognition and determines proper synapse formation may eventually
be answered through the study of these cells and their growing
processes in primary tissue culture.
Use of primary cultures of nervous tissue is a well developed
approach, even with invertebrate tissue. Thus far, however, nerve
cell culturing studies in the molluscs have focused on Aplysia
(Schacher and Proshansky, 1983; Kaczmarek et al., 1979), and no
comparably systematic work has appeared on techniques of squid
neuron culture. Because of the attractive model system presented by
the giant fiber lobe cell bodies and their giant axons for studying Na
channel trafficking, this study was undertaken to begin defining an
effective method for culturing neural tissue from squid, and to
identify some parameters that influence neurite outgrowth.
Materials and Methods
Animals and cell preparation. Egg capsules of Loligo opalescens
collected in Monterey Bay or from spawning in laboratory holding
tanks were kept in tanks with running sea water. 21-24 day old
embryos (Fields, 1965) were pinned through the area immediately
posterior to the arms to Sylgard (DowCorning Corp) and the optic
lobes (including attached central nervous system tissue) and the
stellate ganglia were excised in filtered (0.22um) sea water using
sterile instruments and a Wild stereo microscope. The tissues were
then treated with non-specific protease in filtered sea water
(Img/ml, Sigma Type XIV), for 1 minute, and then washed in filtered
sea water for 3-5 minutes.
Cell culture coverslips and dishes. Coverslips used for culturing
were manually coated with 5 ul of 18 poly-L-lysine (Sigma, MW»
300,000) solution made with distilled water. Coated coverslips
were air-dried for 10-30 minutes (under a dust hood), rinsed with
sterile, distilled water, and then air-dried a second time.
In addition to poly-L-lysine, collagen was also used as a
substrate. Adult squid collagen was obtained from the connective
tissue tunics covering the mantle as described by Gosline and
Shadwick (1983). Blotted tunics (approx. 0.13g total) were added to
10Oml of distilled water, adjusted to pH 2.9 with HCL, and autoclaved
in an effort to obtain collagen in solution. The autoclaved mixture
was then filtered and Iml of it was placed in each culture dish for
about 15 hours, after which time the solution was poured off and the
dishes allowed to air-dry for 30 minutes.
Cell culture plating. A drop of medium (see below) was placed on a
prepared coverslip in a 10 x 35mm plastic petri dish (Falcon). The
previously proteased neural tissue was then mechanically disrupted
with forceps in the drop of medium in order to release neurons for
culturing. Usually one optic lobe or two stellate ganglia were used
per dish. The resulting standard density was from 3-20 cells/0.03
mme. In one dish plated at a higher density the resulting density was
from 15-50 cells/O.03mm4. The cells were then allowed 15-20
minutes to settle after which time 2 ml of medium was added to
each dish. Cells plated in the collagen-coated dishes were allowed 40
minutes to settle. All cultures were maintained at 15.7°C in a
temperature control room. One dish was also kept at 17-21°C using
the standard medium (see below) with no notable difference in
development of processes.
Growth media. The standard L-15-based medium used for squid
neurons contained L-15 (Gibco) plus 6% Fetal Bovine Serun (Hyclone),
12.5 ug/ml chloramphenicol (Sigma), and added salts to final
concentrations of: 10 mM Caci», 10 mM KCI, 50 mM MgCI, and 434.2
mMNacl. Final osmolality was 980 mösm/Kg H9O. The pH was
adjusted to the desired level between 7.4 and 8.0 with HCI and NaoH.
For the experiment using whole squid blood as a medium additive,
blood was extracted from the anterior aorta and cephalic vein of adult
Loligo opalescens. Hemolymph was prepared from whole blood by
centrifugation at 40,000 RPM (100,000g) for 16 hours at 57C. This
resulted in a visually obvious blue pellet (presumably of hemocyanin)
from which the hemolymph could be decanted. Blood and hemolymph
were stored frozen (-20°C).
Culture examination and cell counting Cultures were periodcally
examined with an Olympus IMT-2 inverted microscope (600X) using
phase contrast optics. The 35mm slide field defines a rectangle
(actual size of 210um X 140um with the 40X ob jective used) which
served as a convenient standard area for cell counting. 15-22
pseudo-randomly chosen fields were examined per dish and the total
number of living cells and the numbers of cells showing neurites
counted. Typically, 150-200 cells were counted per dish during each
examination.
Results
Neurons grown in the standard L-15 based medium on a
poly-L-lysine substrate readily grew processes in culture. Mono, bi,
and multi-polar cells from optic lobe cultures are shown in plates i
and 2. Cells typically had somata with a diameter of 5-8 um and
neurite processes of 3-25.5 um in length. In addition, some cells with
neurites appeared to make connections with other cells (plate 2).
Outgrowth of processes occured within hours after plating, and
changes in individual cells over time are shown in plate 3.
Stellate ganglion cells failed to develop processes in culture
during this study, even though both types of neural tissue were
treated identically and cultured in parallel throughout. The remainder
of this paper will describe results on optic lobe neurons in more
detail.
In general, the cells survived in culture without changing the
medium for up to 10 days. Äfter 10 days, many of the cells' interiors
looked darker and therefore the health of the cells was questionable.
One dish had the medium replaced after 5 and 21 hours, but this
revealed no significant influence on neurite development or cell
survival. Effects of medium changes later during culture were not
investigated. Fungi did develop in three dishes after about 5 days and
bacteria in two dishes after four days. Overall, chloramphenicol
seemed to be very effective in controlling contaminations, compared
to penicillin-streptomycin which was used in the earliest stages of
this work.
Effects of pH Squid blood was extracted from an adult animal and
found to have a pH of 7.7, cells were therefore plated and cultured at
this pH. It seems likely that pH would be a very important factor in
squid tissue cultures, because of the strong controll exerted by pH on
aerobic versus anaerobic metabolic pathways in molluscs (Prosser,
1973). The effects of relatively small pH changes around pH 7.7 on
neurons in culture can be seen in Figure 1.
At pH 7.7 neurites develop rapidly, and the percentage of cells
with processes peaks 2-4 hours after plating and starts to slowly
decrease after 24 hours. Cells cultured at pH 7.4 and 8.0 show a
similar time course for the rise and decline of development of
processes, but the maximim number of cells growing neurites can be
affected by small changes in pH. Although it appears that cultures at
PH 7.7 fared better than cultures at either pH 7.4 or 8.0, only the pH
8.0 data showed a statistically significant inhibition of neurite
development (see Table 1). In 3 cultures testing pH 7.7 versus 7.4, no
significant difference in the total percentage of cells developing
neurites was noted. Since pH 7.7 and 7.4 definitely were more
effective, all further experiments were carried out at these values.
Effects of bovine serum vs. squid blood In order to find out how
whole squid blood as an additive might compare with bovine serum,
the 6% bovine serum was omitted from the medium and 107 squid
blood was added instead. As can be seen from Figure 2, the medium
with whole blood (□) was no better than the L-15 based medium with
po serum added at all (A) and not nearly as effective as the standard
medium containing 6% bovine serum (• ). Thus, fetal bovine serum
stimulates neurite growth, whereas squid blood has no detectable
effect.
As a check on whether there might be something in squid blood
which actually hindered neurite growth, the whole blood was
centrifuged (see methods) which presumably removed most of the
hemocyanin which is present in the blood at 80 mg/ml (Prosser,
1973). Even this hemolymph without the hemocyanin (—) produced no
positive effect as a medium additive. Because the hemolymph and
whole blood produced very similar results, it seems that the absence
or presence of hemocyanin cannot be the sole factor responsible for
the results in Figure 2. Finally the addition of whole blood to the
standard medium containing 6% bovine serum (m) appears to reduce
the efficacy of the standard with bovine serum as an additive.
Effect of substrate. It has been shown that a collagen substrate
is necessary for cell differentiation in chick skeletal muscle cell
cultures (Work, and Burdon, 1980), and collagen is present in large
amounts in squid connective tissue and mantle (Gosline and Shadwick,
1983). Therefore, collagen was investigated as an alternative to
poly-L-lysine. The preparation of squid collagen is described in the
methods section, mammalian collagen was not tried. Cultures plated
on the collagen grew processes very poorly compared to cultures
plated on poly-L-lysine (see Figure 3 and Table 1).
Effect of cell density. A density study was carried out because of
the possibility that cells might condition the medium or influence one
another through contact in a positive or negative manner. The results
suggest that the cells tend to develop more effectively in the
standard density of 3-20 cells/O.03mm4 rather than the higher
density (see Methods) of 15-50 cells/O.03mmé. The more dense
culture plated with the standard medium, pH 7.4, reached a maximum
percentage of cells with processes of only 38 whereas cells plated
at the standard density in the same medium reached a maximum of
127 (pH 7.4, Figure 1).
Discussion
In summary, the pH of the cultures conclusively influences the
growth of processes from neural cells in culture, with pH's of 7.4 and
7.7 proving the most effective. Further, the use of bovine serum in
the dishes appears to be necessary for growth of processes and more
effective than whole squid blood or hemolymph. The addition of squid
blood to the culture with bovine serum seems to have negative
effects. Finally, the presence of hemocyanin, the changing of the
medium at 5 and 21 hours, and the growing of cells at room
temperature do not seem to effect the cells in culture to any notable
degree.
The results from using collagen as a substrate rather than
poly-L-lysine may be incorrectly interpreted as saying that collagen
does not promote the development of processes. However, it seems
more likely that these results point to a problem in the method of
collagen preparation.
The development of neurites appears to be hindered by the
addition of squid blood to the medium with bovin serum. Here, it is
interesting to note that in the development of the squid embryo, the
nervous system develops from day 9 to about day 18 whereas the
hearts begin to beat at about 21-24 days (Fields, 1965). Perhaps the
earlier development of processes in the animal is inhibited by the
blood.
Although a brief examination of changing the medium was
carried out, further study should be completed to see if changing the
medium every 24 hours for several days might help maintain the
processes in culture for a longer period of time.
The results of the density study should be taken as an obsrvation of
the density which seems to be most beneficial to the cultures. In
10
addition, it may serve as incentive for a further density studies with
a much larger sample size.
Finally, it is possible that the stellate ganglion never developed
processes in culture because they are too far along in their
development at the later embryo stages. Therefore, it may be
interesting to use ganglion from earlier embryo stages although the
dissections may prove more difficult.
Further directions of study may include answers to such
questions as: does pH effect process length, branching, or number of
processes? Does the presence of an original process effect the
initiation of growth or restrict the number of processes? These are
some factors that have been shown to be influenced by the presence
of hemolymph in Aplysia neurons in cell culture (Schacher and
Proshansky,1983).
Literature Cited
Fields, G. (1965) The Structure, Development, Food Relations,
Reproduction, and Life History of the Squid, Loligo opalescens Berry.
(The resources agency Dept. of Fish and Game, CA).
Fuchs, P. et al (1981) Membrane Properties and Selective Connexions
of Identified Leech Neurons in Culture: J. Physiol. 316: 203-223.
Gosline, J. and R. Shadwick (1983) Molluscan Collagen and Its
Mechanical Organization in Squid Mantle: The Mollusca. 1: 371-396.
Kaczmarek et al, (1979) The Morphology and Coupling of Aplysia Bag
Cells within the Abdominal Ganglion and in Cell Culture: J. Neurobio.
vol 10, no. 6: 535-550.
Prosser, C. Ed. (1973) Comparative Animal Physiology (Saunders
college pub., Philadelphia) pp. 218-220 and 327.
Schacher, S. and E. Proshansky (1983) Neurite Regeneration by Aplysia
Neurons in Dissociated Cell Culture: Modulation by Ap/siø Hemolymph
and the Presence of the Initial Axonal Segment. J. Neurosci. 3:
2403-2413.
Work, T. and R. Burdon, Eds. (1980) Cell Culture for Biochemistry and
Molecular Biology. (Elsevier/North-Holland and Biomedical Press, NY)
p. 78.
Young, J. (1939) Phil. Trans. R. Soc. London Ser. B229, 465-503.
Culture condition
Standerd pH 7.4
PH 7.
PH 8.0
Collagen pH 7.4
Hemolymph pH 7.4
No. dishes
2
Table 1
Maximum
with processes (mean) 1/ std. dey
15.
2.3
6.1
22
3.1
2.1
2.8
Figure Legend
Figure 1. pH influence on neurite outgrowth. The total percentage of
cells with processes during time in culture are plotted for 3 pH
values as indicated. All dishes use the standard media on
poly-L-lysine. The 7.7 and 7.4 curves represent 3 dishes each
whereas the 8.0 curve represents 2 dishes. The significance of the
apparent differences can be seen in Table 1. Cells at pH 7.7 and 7.4
did significantly better than at pH 8.0.
Figure 2. Comparison of fetal bovine serum with squid blood as a
medium additive. Percentages of cells with processes over time in
culture is indicated for the following additives
(1) () L-15 plus 6% serum (3 dishes).
(2) (m) L-15 plus 6% serum plus 10% whole squid blood (1 dish).
(3) (A) L-15 only (1 dish).
(4) (») L-15 plus 10% squid hemolymph (1 dish).
(5) ( L-15 plus 10% whole squid blood (1 dish).
All dishes are at pH 7.7, plated on poly-L-lysine. Although not
statistically analyzed, it can be seen from the graph that: there is an
inhibition of process growth if serum is omitted from the medium.
Replacing serum with 10% squid blood or 103 hemolymph drastically
hinders process development. The presence (as in whole blood) or
absence (as in hemolymph) of hemocyanin does not alter this result.
Addition of 10% squid blood to L-15 plus 6% serum may also inhibit
the development of processes, but the effect is not so clear cut.
Figure 3. Using collagen as a substrate rather than poly-L-lysine.
Both groups use the standard media at pH 7.4. Two cultures were
plated on collagen and three on poly-L-lysine. The collagen culture did
significantly worse than the poly-L-lysine cuture (see Table 1).
Table 1: Comparison of maximum 3 of cells with processes,
statistically analyzed with an unpaired t-test. pH's 7.7 and 7.4 both
had significantly higher max. % with processes than pH 8.0. Further,
the pH of 7.4 did significantly better on poly-L-lysine than collagen
and with bovine serum rather than hemolymph.
Plate 1: Optic lobe cells with relatively simple neurite processes.
a-c) show mono-polar cells d,e) give examples of bi-polar cells. All
cells are from cultures with standard media:
a: pH 7.4, 23 hrs after plating.
d: pH 7.4, 48 hrs.
b: pH 7.7, 9.25 hrs.
e: pH 8.0, 24.5 hrs.
C: pH 7.4, 54 hrs.
Plate 2: Optic lobe cells with extensively developed processes. a,b)
are examples of multi-polar cells. c-e) are cells that appear to make
connections with each other. All cells are from cultures with
standard media:
a: pH 7.7, 9.25 hrs. after plating.
C: pH 7.7, 9.25 hrs.
d: pH 7.4, 54 hrs.
b: pH 7.4, 54 hrs.
e: pH 7.7, 24 hrs.
Plate 3: Time-lapse pictures of cells developing processes. a,b)
shows several cells in standard media (pH 7.7) 0.5 hrs after plating
and 3.5 hrs after plating. c-e) Example of one cell developing, from
the same culture, 17.75, 18.75, and 19.75 hrs after plating
(respectively).

+ 4
V

Acknowledgements
It is with great gratitude that I thank the entire Hopkins Marine
Station staff for making Bio. 175H such a success. In particular,
would like to thank Judy Thompson for holding the whole program
together; Chuck Baxter, Mark Denny, and Stuart Thompson for their
encouragement, interest, and advice. A very special thank you to
William Gilly for his persistence in challenging students' ideas,
preconceptions, limitations, and fishing skills, and to Patricia
Gosling for her seemingly unlimited patience and many hours of help.