GFAP Immunoreactivity in Octopus Nervous System
Before and After Injury
Abstract
Astrogliosis following injury to the mammalian central nervous
system is accompanied by increased synthesis of glial fibrillary
acidic protein (GFAP) and the lack of regeneration. In order to
extend studies of GFAP expression in relation to injury in taxa of
higher regenerative capacity, we chose Octopus rubescens as a model
system. We used a polyclonal anti-human GFAP in conjunction with
immunoblotting and immunohistochemistry techniques applied to optic
lobes of the brain and to peripheral axial nerve cords in the arms.
Animals were also studied before and after lesion to the axial
nerves that contain ganglionic regions of neuronal and glial
somata. In uninjured Octopus,
Western blots with boh tissue
sources revealed a single protein band (140KD). Pre-absorption of
the antibody with purified human GFAP eliminated this band, which
is larger than the 48-51 KD
protein found in vertebrates.
Immunostaining of fixed and sectioned material from axial nerve
cords showed that anti-GFAP preferentially stained cellular
regions. This pattern is distinct from that produced by anti¬
neurofilament, which stained primarily neuropil and axons. Studies
were also carried out following injury (amputation) to the axial
nerve cords. Nine days after injury, immunoblotting revealed an
increased level of the 140 KD band. Experiments on other
cephalopods were also carried out. In uninjured Sepia officianalis,
Sepioteuthis lessoniana, and Loligo opalescens, the 140 KD band was
not detected by Western blots with optic lobe proteins. This
product was obtained, however, with axial nerve material from
Sepia. These results (i) indicate the presence of a GFAP-like
protein in the nervous system of cephalopods, (ii) show that
increased protein levels accompany injury in the peripheral nerve
cords in Octopus, and (iii) suggest that the protein may be
associated with glial cells.
Introduction
In mammalian systems, there is experimental evidence that
glial scarring presents an impediment to regeneration in the
central nervous system (Reier et al., 1983; Windle et al., 1952).
Such glial scarring is characterized by swelling of the astrocyte
cell bodies and extension of the processes. This hypertrophy of
astrocytes following injury, termed astrogliosis, is accompanied by
increased synthesis of glial fibrillary acidic protein (GFAP) (Eng.
1988). In most cases, regenerating axons are unable to penetrate
the scar tissue. Although the accumulation of GFAP in these
reactive glial cells appear to inhibit regeneration in mammalian
CNS, they do not seem to present an obstacle to regenerating axons
in lower vertebrates such as the teleosts (Stafford et al., 1990:
Anderson et al., 1984).
In invertebrates, the ability to repair damage to the nervous
system surpasses that of the vertebrates (Fredman and Nutz, 1988).
However, the roles of glial cells in response to injury in these
model systems have not been examined extensively. The lack of glial
markers for immunochemistry studies and the limited knowledge of
glial cells in these organisms beyond morphology make these studies
difficult.
In the present study, we investigate a glial filament protein
in cephalopods that appears to be related to GFAP in vertebrates.
In a previous report, it was found that a monoclonal antibody to
GFAP cross reacted with three bands (56, 49, 37 KD) in protein
blots of Octopus vulgaris optic lobe homogenate. Using
immunoelectron microscopy, it was found that the antibody labelled
filaments that may be glial in nature (Cardone and Roots, 1990). In
this study, we use a polyclonal antibody to study GFAP
immunoreactivity in normal and injured axial nerve cords from
Octopus rubescens, which are able to regenerate these nerve cords.
These tissues basically consist of a series of ganglia
arranged along the arms and embedded within the musculature of the
arms. In each ganglion, there is an outer cellular layer that
surrounds the neuropil (Young, 1971). Immunohistochemistry
techniques were utilized to examine if GFAP immunoreactivity was
restricted to certain regions of the tissue.
We also carried out comparative studies of GFAP
immunoreactivity in other uninjured cephalopods, Loligo opalescens,
Sepioteuthis lessoniana, and Sepia officianalis.
Materials and Methods
ANIMAL PREPARATION:
Octopi (Octopus rubescens) about 8 inches in arm span were
collected in Monterey Bay, California. They were kept in holding
tanks with circulating sea water and fed small live crabs. For
injury experiments, the animals were anesthesized in 23 ethanol
until arm movements stopped (about 5 to 10 minutes), and four of
the arms were amputated about 3 cm from the tip. The animals were
placed back in the holding tanks. Nine days after amputation, axial
nerve cords were removed from the proximal stumps and control arms
for immunoblotting.
For comparative studies of GFAP immunoreactivity in other
cepahalopods, Loligo opalescens, Sepioteuthis lessoniana, and Sepia
officianalis were used, L. opalescens as collected in Monterey Bay,
Ca.; S. lessoniana as provided by Dr. Roger Hanlon at the
University of Texas, Galveston, Medical Branch; and S. officianalis
as donated by the Monterey Bay Aquarium. All three were kept in
tanks with circulating sea water. The nervous system tissues used
for immunoblotting and immunohistochemistry were removed from these
animals following decapitation without anesthesia.
IMMUNOBLOTTING:
Nervous system tissues were removed from the animals and
placed directly in phosphate buffer (pH 8.0) containing 13 SDS, 2
mM EDTA, and 3 mM EGTA. The samples were homogenized in a motor
driven homogenizer, centrifuged at 12,000 rpm (Eppendorf microfuge)
for 30 minutes, and the supernatant collected. Protein
concentrations were determined by the bicinchoninic acid (BCA)
assay (Brown et al., 1989). The proteins were separated using 103
polyacrylamide gel electrophoresis according to the method of
Laemmli (1970) and transferred onto nitrocellulose membrane (Towbin
et al., 1979). Antibody detections were done on the blots using a
previously described polyclonal rabbit antiserum to human GFAP (Eng
and DeArmond, 1983), monoclonal mouse antiserum to neurofilament
(Sternberger and Sternberger, 1983), and the peroxidase anti¬
peroxidase method (PAP) (Sternberger et al., 1970). To test
specificity of staining, the same rabbit anti-GFAP pre-absorbed
with purified GFAP was obtained from Dr. Lawrence Eng, Department
of Pathology, Stanford University Medical School.
Nonspecific binding was minimized by preincubation with normal
swine serum for polyclonal or normal goat serum for monoclonal; and
all incubations were done in 18 milk in buffer. The blots were
incubated in primary antibodies overnight at a dilution of 1:500.
After incubation in the secondary antibody and the PAP complex,
cross-reacting proteins were visualized by development in 3,3'-
diaminobenzidine.
IMMUNOHISTOCHEMISTRY:
Tissues were removed from animals and fixed in 43
paraformaldehyde at 4'C for 48 hours. Paraffin sections of these
tissues were prepared by standard techniques (Galigher and Kozloff,
1964). Briefly, tissues were dehydrated through graded alcohol,
alcohol replaced with xylene, and tissues gradually infiltrated
with melted paraffin. The infiltrated tissues were then embedded in
paraffin and sectioned at 10 microns with a steel knife microtome
and mounted on albumin coated slides. Prior to immuostaining, the
slides were deparaffinized in xylene, rehydrated through graded
alcohol, and treated with acetylated Trypsin (Sigma Chemicals) and
hydrogen peroxide. Immunostaining was done using the same
antibodies as described above and the PAP method. The sections were
then counterstained and mounted for viewing.
Results
IMMUNOBLOTTING:
The polyclonal anti-GFAP cross reacted with one band (140 KD)
in transblots of optic lobe and axial nerve cords from uninjured
Octopus (fig. la). This protein is of higher molecular weight than
mammalian GFAP (48-51 KD). However, using the same antibody pre¬
absorbed with the purified mammalian antigen, the band was
eliminated (data not shown). To further demonstrate that the
antibody was not directed to neurofilament protein (NF), the same
transblots were immunostained with a monoclonal anti-NF. NF
staining revealed bands clustered around 200 KD and one strong band
at 110 KD, none of which corresponded with the GFAP band (fig. 1b).
INJURY EXPERIMENTS:
Nine days after amputation of the arms, there were no visible
signs of regeneration. However, the stumps were still active,
responsive to touch, and showed no signs of degeneration. The axial
nerve cords from injured and control arms removed at this point
showed differing levels of GFAP immunoreactivity. From the
transblot stained with anti-GFAP (fig. 2a), it appears that the 140
kD band is more intense in the injured tissue than in the control.
This difference is evident despite the greater amount of protein
loaded for the control tissue (fig. 2b). This rise in the level of
GFAP immunoreactivity is accompanied by the appearance of another
cross reactive band at 80 KD. However, the significance of this
band in relation to injury is questionable since the band sometimes
appears in uninjured tissues as well (data not shown).
IMMUNOHISTOCHEMISTRY:
Immunostaining of paraffin sections of Octopus axial nerve
cords and Sepioteuthis stellate ganglia with anti-GFAP and anti-NF
showed different patterns of staining. With anti-GFAP, staining was
more diffuse. However, it appears that staining was primarily in
layers rich in cell bodies, in particular the interganglionic
regions (fig. 3a). In Sepioteuthis stellate ganglion, the antibody
stained material which seemed to surround and adhere tightly to the
large neuronal cell bodies (fig. 4a). In contrast, anti-NF stained
the cytoplasm of these cell bodies (fig. 4b). In sections of
Octopus axial nerve cords, anti-NF stained mostly the neuropil and
nerves (fig 3b).
GFAP IMMUNOREACTIVITY IN CEPHALOPODS:
Immunoblotting was also carried out using tissue homogenates
from other uninjured cephalopods. We were able to detect the 140 KD
protein in the axial nerve cords of Sepia. Results with Loligo
stellate ganglion were not conclusive, probably because of an
insufficient amount of tissue to work with. The results of
experiments on various tissue sources are summarized in figure 5.
We were not able to detect this protein in the central nervous
systems of the cuttlefish or squid; this is in contrast to the case
in Octopus.
Discussion
The level of differentiation and specialization of glial cells
tends to be a function of the complexity of the nervous system of
an organism (Cardone and Roots, 1990). At the level of cephalopods,
the development of the gliovascular system (Young, 1971) is an
indication of this trend of specialization and differentiation. In
vertebrates, the glial cells have become highly specialized cells
in function and structure. Although little is known about the
properties of glia in cephalopods, the fibers that support the
processes of the glial cells as described by Young (1970) may be
related to glial filament proteins in mammals.
GFAP has been shown to be the major structural protein in
differentiated astrocytes (Eng, 1985). Using a polyclonal rabbit
antiserum to human GFAP and immunochemistry techniques, we have
shown cross reactivity of the antibody to a specific protein in
Octopus central and peripheral nervous systems, as well as parts of
the peripheral nervous system of other cephalopods. Furthermore,
using immunohistochemistry, we were able to show that this protein
does not co-localize with neurofilament protein, an indication that
the protein may be associated with glial cells. The product we
obtained was a 140 kD protein. This is unusual as variations in the
molecular weight of mammalian GFAP (48-51 KD) are small (Dahl and
Bignami, 1973). The size of the Octopus protein we obtained
conflicts with the findings of Cardone and Roots, who detected
three protein bands in Western blots with a monoclonal anti-GFAP
(56, 49, and 37 KD). It is interesting that the molecular weights
of the three protein bands sum up to approximately 140 KD. Although
the reducing conditions of the gel sample buffer were similar in
both studies, it is still possible that (i) the high molecular
weight we obtained may be the result of aggregation of the three
lower molecular weight bands, or (ii) the three smaller bands may
be degradation products of the larger protein we found.
To draw further connections between this 140 KD protein and
vertebrate GFAP, there appears to be an increase in expression
following injury. The role of GFAP in glial scarring is well
established in vertebrates (Eng, 1988; Reier et al., 1983). This
increase in the level of GFAP immunoreactivity may constitute a
similar event in the Octopus. Although in the present study we did
not wait long enough for the arms to regenerate, observations have
confirmed that the arms are able to do so (Cousteau and Diole,
1973). Thus it seems that the increase in this GFAP-like protein
expression does not present an obstacle to neural regeneration.
It is possible that the high regenerative capacity of the
nervous system of the Octopus may be related to the high molecular
weight GFAP. In central nervous system tissue from cephalopods of
presumably lower regenerative capacity, Sepia, Loligo, and
Sepioteuthis, we could not detect the 140 KD protein by
immunoblotting. In the peripheral arm nerves of the Sepia and the
peripheral stellate ganglion of the Sepioteuthis, however, we were
able to see cross reactivity with the antibody.
Thus, there may be a link between this GFAP-like protein and
vertebrate GFAP beyond shared antigenic properties. Structural and
biochemical similarities may be reflected in functional
similarities as well.
Acknowledgements
This work was made possible with valuable contributions from
Dr. W. F. Gilly. We thank the laboratories of Dr. D. Epel and Dr.
D. Powers for the use of equipment, Y. L. Lee for valuable advice,
Bruce Hopkins for assistance in collecting animals, and Chris
Patton for expertise in photography of the blots. The antibodies
were generous gifts from Dr. Lawrence F. Eng.
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B. GFAP NE
A.
200-
97.4-
69-
46-
30-
2 3 45
Figure 1. (a) GFAP
staining of protein blot of Octopus
left optic lobe
(lane 1); right optic lobe (lane 2);
axial nerve cord
(lane 3); rock fish (Sebastes mystinus
spinal cord (lane 4); mouse brain (lane 5).
(b) GFAP and NF
staining of protein from axial nerve
cord of Octopus.
The GFAP band does not correspond with
any of the bands stained by anti-NF.
A. Anti-GFAP
B. Total Protein
200-
97.4-
69-
46-
30
2
Figure 2. (a) GFAP staining of proteins
from Octopus axial nerve cords after
injury (lane 1) and before injury (lane
(b) Coomassie stained total protein from
(a) show lower amounts of protein loaded
for the injured tissue and the bands
corresponding to the GFAP bands.
Figure 3. (a) GFAP staining of longitudinal
section of Octopus axial nerve cord showing
strong staining in the interganglionic region
(arrows) and the cellular layer. This section
was not counterstained.
(b) NF staining of the same tissue with
staining strongest in the neuropil.
Figure 4. (a)
GFAP
staining of Sepioteuthis
stellate ganglion showing staining of possible
glial cells that extend processes to wrap
around the large neuronal cell bodies (arrows).
(b) NF
staining of
same section showing
staining of the cytoplasm of the cell bodies.
GFAP IMMUNOREACTIVITY IN VARIOUS
CEPHALOPOD NERVOUS SYSTEM TISSUE SOURCES
Brain
Optic
Stellate
Axial
Lobe
Ganglion
Nerve
Cord
Octopus rubescens
Sepia officianalis
Loligo opalescens
+ ?
Sepioteuthis lessoniana

Figure 5.