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5
Marine Organisms as Models for Biomedical Research
Ought we, for instance, to begin by
discussing each separate species…taking each kind in hand
independently of the rest, or ought we rather to deal first with
the attributes which they have in common in virtue of some common
element of their nature, and proceed from this as a basis for the
consideration of them separately? (Aristotle, De partibus
animalium).
Recognition of the conservation of fundamental processes during
evolution requires the comparative study of many different species.
As indicated by the quotation from Aristotle introducing this
chapter, there is a long tradition behind this comparative approach
to biology. The analysis of conserved features has been essential
to the study of evolution and the reconstruction of evolutionary
relationships. Although the comparative approach emerged from the
tradition of natural history, it has also been used extensively in
the disciplines of physiology, biochemistry, and developmental
biology. Some of the insights gleaned from these studies include
the thermoregulatory role of countercurrent exchange systems in the
circulatory system, the biochemical evolution of proteins through
duplication of structural motifs, and the role of cytoplasmic
segregation in the development of embryos. Comparative studies have
also helped researchers identify which features of organisms are
fundamental to function. The assumption is that a conserved feature
is so essential for normal biological processes that any
modification would be likely to reduce the viability of the
organism.
Even after a critical function has been identified, studies
using a diversity of organisms have facilitated the elucidation of
mechanisms. Determining the best method for investigating a
particular biological phenomena frequently requires choosing the
animal model that best lends itself to experimentation. For
example,
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the characterization and cloning of the acetylcholine receptor
was simplified by the use of the electric organ, a highly modified
muscle found in the electric ray Torpedo. Nerve impulses are
chemically transmitted to muscles through the acetylcholine
receptor, a process disrupted in the human neuromuscular disorder,
myasthenia gravis. In the ray's specialized electric organ, this
receptor protein is found at such a high density in the cell
membrane that researchers were able to determine the structure of
the protein and clone the gene without first having to purify the
protein. This would not have been possible using human or other
mammalian tissue. The validity of applying results obtained from
the study of a protein in a highly modified fish muscle to normal
biochemical processes in human muscle derives from the insight that
many fundamental features at the molecular and cellular level are
highly conserved even though the evolution of animals has shown
dramatic changes in morphological form (Gerhart and Kirschner,
1997).
Studies using marine organisms have had a major influence on
biomedical research (Sargent, 1987). This chapter highlights some
of the best recognized marine models and elaborates the reasons for
their success. However, the first question one might ask
is—why marine organisms? At higher taxonomic levels, most
biological diversity is found either primarily or exclusively in
the ocean. Of 33 modern phyla, only 11 are found in terrestrial
habitats while 28 occur in marine habitats. Hence the diversity of
life in the sea offers more possibilities for the discovery of
organisms for use as models to explore various biological
processes. In several of the examples described in this chapter,
specific adaptations to the marine environment have been valuable
in studying analogous physiological processes in humans. Of
particular interest are several marine taxa that share a common
origin with mammals. This group, the deuterostomes, includes
vertebrates (and other chordates), echinoderms (e.g., sea urchins
and sea stars) and tunicates (e.g., sea squirts). Echinoderms
appeared early in the fossil record and are the most distant
deuterostome relatives of humans. Studies on the differences and
similarities of these groups offer insights into the evolution of
vertebrates and mammals. Also, many of these organisms have
specialized features that have enabled researchers to elucidate
complex processes that would be more difficult to study in mammals.
A number of examples are listed in Table 5-1, of which some are
described in more detail in this chapter.
Sea Stars, Sea Urchins, Tunicates, and
Sharks:
Their Role in Understanding How the Body Fights Infection and
Disease
In 1882, Elie Metchnikoff conducted an experiment with sea star
larvae (Beck and Habicht, 1996). He punctured a larva with the
thorn of a rose and the next day observed tiny motile cells
surrounding the thorn. He postulated that the motile cells were a
defense mechanism against foreign invaders. The process
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TABLE 5-1 Examples of Marine Species Used in
Biomedical Research
General Human Health Issue
Taxon
Species (Common Name)
Human Medical Concern
Immunology
Mollusca
Conus spp. (cone snails)
Blood disorders, clotting, hemophilia
Echinodermata
Arbacia punctulata (sea urchin)
Cell-mediated immune system responses
Tunicata
Botryllus schlosseri (Golden Star Tunicate,
sea squirt)
Immune systems and disorders (self/non-self
recognition), AIDS/HIV transmission
Vertebrata, Fish
Squalus acanthias
(spiny dogfish shark)
Immune system function, evolution of antibodies,
and disease resistance
Neurobiology
Mollusca
Loligo pealei (squid) Aplysia
(marine snail)
Neurological studies, behavior Nerve impulse
transmission
Arthropoda
Limulus polyphemus (horseshoe crab)
Vision Neural basis of behavior
Vertebrata, Fish
Opsanus tau (toadfish)
Balance and equilibrium, nausea
Squalus acanthias (spiny dogfish shark)
Brain function Vision, glaucoma, cataracts
Pomacentrus partitus (bicolor
damselfish)
Neurofibromatosis
Electrophorus electricus (electric eel)
Synaptic transmission (NA+ K+ ATPase)
Cell Biology/
Cancer
Mollusca
Spisula solidissma (surf clam)
Cell division/cancer
Loligo pealei (squid)
Cell physiology, intracellular transport, and
cellular pH calcium regulation
Echinodermata
Arbacia punctulata (sea urchin)
Cell division/cancer
Fertilization and development
Arthropoda
Cancer irroratus (red crab)
Organic ion transport
Vertebrata, Fish
Pseudopleuronectes americanus (winter
flounder)
Organic ion transport
Physiology
Arthropoda
Eriocheir sinensis
(Chinese mitten crab)
Cellular osmoregulation
Cancer irroratus
(red crab)
Detoxification mechanisms
Carcinus maenas
(green shore crab)
Kidney function
Vertebrata, Fish
Opsanus tau (toadfish)
Insulin secretion and diabetes
Muscle pathologies
Squalus acanthias
(spiny dogfish shark)
Cystic fibrosis Kidney and heart research
Pseudopleuronectes americanus (winter
flounder)
Detoxification mechanisms
Anguilla rostrata
(American eel)
Kidney function
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Representative terms from entire chapter:
sea urchin
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Metchnikoff observed was phagocytosis. Although phagocytosis had
already been observed with human cells, his observations led him to
suggest that the process might be a more fundamental defensive
mechanism that is widespread in the animal kingdom. Further
research showed that echinoderms (e.g., sea urchins and sea stars)
possess the features of a basic immune system, one that involves
the non-specific action of phagocytic cells (Smith and Davidson,
1994). This is believed to be the oldest form of immunity and, as
suspected by Metchnikoff, it appears to be shared by all animals.
Metchnikoff's work on sea stars laid the foundation for the
disciplines of cellular and comparative immunology and all
subsequent studies on the role of cells and phagocytosis in
fighting infection and disease in humans.1
Further insight into how the human immune system functions has
been obtained from studies of other marine deuterostomes.
Tunicates, or sea squirts as they are commonly called, have
provided a model for studying another aspect of immunity, the
ability to distinguish ''self" from "non-self." When two sea
squirts come into contact they will fuse into one organism if
related or grow apart if they are unrelated. Observations of this
phenomenon in the field led to the laboratory use of tunicates as a
model system for tissue transplantation studies (Raftos, 1994).
Tissue recognition or rejection is mediated by the immune system.
In sea squirts and humans, similar strategies have been described
for determining tissue compatibility involving specialized cells
and specific self-recognition molecules.
The recognition of "self" is also important to the reproduction
of sea squirts. These animals are hermaphrodites, meaning that the
same individual can produce both sperm and eggs. However, there is
no fertilization of eggs by sperm produced from the same animal.
Studies of the tunicate Botryllus schlosseri also showed
that the sperm from one individual does not bind to blood cells
from the same individual, although they do bind to blood cells from
a different individual. This observation prompted some AIDS
researchers to conduct a similar experiment with human sperm and
blood cells (Scofield, 1997). They found that human sperm, like
tunicate sperm, exclusively bind to blood cells from other
individuals and discriminate through detection of a
self-recognition molecule on the surface of the blood cells
(Scofield et al., 1992). This discovery may play a role in
understanding how the AIDS virus is transmitted and help biomedical
researchers devise protocols for reducing disease transmission.
The phagocytic cells of marine invertebrates constitute the most
fundamental type of animal immune system. The response is rapid and
is referred to as natural or innate immunity. Vertebrates possess
an additional form of immunity referred to as acquired or adaptive
immunity. This form of immunity relies on a combinatorial genetic
mechanism that generates millions of specific recognition molecules
in specialized defense cells, the B and T lymphocytes. Insight into
the evolution
1 The
importance of Elie Metchnikoff's work was recognized in 1908 when
he was awarded the Nobel Prize for Physiology or Medicine.
Page 87
of this important feature of the human immune system has come
from the study of sharks. Sharks and other cartilaginous fishes are
the most primitive group of vertebrates with this adaptive,
combinatorial immune system. Sharks first appeared in the fossil
record between four and five hundred million years ago and their
long history suggests that this "new" immune system gave sharks an
evolutionary advantage which allowed them to survive while other
taxa became extinct (Litman, 1996). The immune system of sharks
shares some similarities to the human fetal immune system because
the predominant circulating class of anti-bodies in the shark
resembles the earliest produced immunoglobulin M (IgM)
macroglobulins of fetal humans. Sharks also possess innate
anti-microbial anti-bodies, T-cell receptors, and major
histocompatability antigens (MHCs). Thus, sharks present a
comparative model for studying both innate and acquired immunity
and autoimmunity, which is the underlying cause of several human
diseases such as lupus and rheumatoid arthritis. In addition,
sharks possess the steroid squalamine which has an immunomodulatory
function and antimicrobial activity with pharmaceutical potential
(Moore et al., 1993). Although many aspects of the immune system
can be studied in mammals, studies that have taken a comparative
approach have provided valuable insight into the basic mechanism of
immune responses. The comparative approach may hold the key to
developing new therapies for autoimmune and immunodeficiency
diseases (Marchalonis and Schluter, 1994).
Sea Urchin and Clam Eggs:
Their Role inUnderstanding Cell Biology and Biochemistry
Sea urchins have served as experimental models for more than 100
years. Many species produce tremendous quantities (millions to
billions; Plate XV) of large, clear eggs that lack external
coatings. An early example of their utility was Otto Warburg's
demonstration in 1908 of the increase in oxygen consumption that
occurs following fertilization, despite the relative insensitivity
of his methods.
The cell cycle is the orderly sequence of events in which a cell
first reproduces its genetic material and then divides. In the life
of an organism, cell division begins following the fertilization of
the egg, defines the early growth and differentiation of the
embryo, and continues throughout adulthood, especially in tissues
like blood and intestinal mucosa. The cell cycle is closely
regulated by a group of proteins, the cyclins (Pines, 1996), which
were originally identified in sea urchins (Evans et al., 1983). The
key features of sea urchin eggs that made this discovery possible
are their abundance and the synchronous division of cells after
fertilization. Researchers radiolabeled newly fertilized eggs to
tag proteins synthesized during the first few cell divisions. They
found that while most proteins accumulate through succeeding cell
cycles, one protein, cyclin, is remarkable in that it is
synthesized and destroyed once per cell cycle, appearing and
disappearing periodically as the cell divides.
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Cyclin A was first cloned from the surf clam and its connection
to the cell cycle was confirmed by its ability to initiate meiosis
in frog eggs and mitosis in somatic cells. Subsequent studies have
shown that the synthesis and destruction of cyclins are the key
events in the regulation of cell division in all eukaryotic cells,
from yeast to human.
Clued by the sequences of the cyclins discovered in marine
invertebrates, scientists have identified many different cyclins in
mammalian cells. Currently, there is considerable excitement among
cancer researchers following the finding that in many human cancers
there are mutations that change the function of particular cyclins
or the proteins that either regulate or are regulated by cyclin
(Hall and Peters, 1996). Thus, the discovery of cyclins in sea
urchin eggs revolutionized the study of the mammalian cell cycle
and paved the way for new research into the diagnosis and treatment
of cancer.
Transient and localized changes in intracellular calcium
concentration are ubiquitous signals for many essential cellular
responses (Lee, 1997). Sea urchin eggs are the preferred model for
investigating calcium signaling because: (1) the eggs are large,
transparent, and amenable to microinjection, allowing localized
changes in calcium concentration to be readily visualized using
dyes and (2) the abundant cytoplasm can be harvested and
fractionated to identify the interacting cellular components of the
signaling pathway.
More than 50 years ago a spike in intracellular calcium
concentration was identified in Arbacia eggs following
fertilization. This calcium signal triggers formation of a clear
proteinaceous fertilization envelope that surrounds the entire egg,
and by preventing sperm from reaching the egg membrane, acts as a
mechanical block to polyspermy. The calcium signal also contributes
to the signal to activate protein and DNA (deoxyribonucleic acid)
synthesis at the onset of development. Curiously, the calcium
signal occurs as a wave that begins at the site of sperm-egg fusion
and sweeps across the entire egg in approximately 30 seconds (Plate
XVI). Such waves, which can be repetitive, occur in many different
kinds of cells in response to a wide variety of stimuli. The nature
of the waves has been elucidated by the discovery that they depend
on chemical intracellular messengers, some of which were only
recently identified in an exciting series of investigations using
sea urchin eggs (Lee, 1997).
The calcium that constitutes these waves is released locally
from intracellular stores identified as the endoplasmic reticulum.
The initial signal, calcium-induced calcium release, propagates the
waves by successively stimulating calcium receptors in adjacent
intracellular membranes, resulting in further calcium release.
Based on new results using sea urchin eggs, there are at least two
separate calcium stores, each with a specific receptor selectively
sensitized by one of two novel endogenous chemicals, cyclic
adenosine 5'-diphosphate-ribose (cADP-ribose) and nicotinic acid
adenine dinucleotide phosphate (NAADP). The cADP-ribose and NAADP
were identified by analysis of sea urchin egg cytoplasm.
Page 89
Additional proteins that modulate cADP-ribose and NAADP activity
were also identified in the egg cytoplasm.
The importance of these findings is highlighted by the discovery
that numerous types of mammalian cells are responsive to
cADP-ribose, including neurons and muscle cells in heart,
intestine, and skeletal muscle. The cADP-ribose acts on ryanodine
receptors that couple increases in intracellular calcium
concentration to muscle contraction. Cyclic ADP-ribose also
accounts for the calcium-mobilizing action of the nitric oxide
signaling pathway.
Thus, the cADP-ribose system that was only recently discovered
as a result of continuing investigations of sea urchin eggs appears
to be of fundamental importance for mammalian neuromuscular
coordination and, with further research, should contribute to
advances in the diagnosis and treatment of neuromuscular
disorders.
Marine Organisms:
Their Role in Physiological Studies Pertaining to Fluid and Ion
Transport,
Renal Function, and Volume Regulation
Marine organisms have proven the value of the August Krogh
principle, which essentially states that for every problem in
physiology, there is one animal ideally suited to solve that
problem. In particular, marine invertebrates and fishes have been
important as models for osmoregulatory phenomena such as fluid and
ion transport, renal function, and volume regulation. It is
important to recognize why marine organisms have developed
extensive osmoregulatory capabilities in order to appreciate how
the principles learned from studying osmoregulation in marine
animals have led to an increased understanding of osmoregulatory
phenomena in humans. Simply, osmoregulation is necessary for the
survival of animals in salt-variable environments ranging from
estuaries and mangrove swamps to the Dead Sea. Marine invertebrates
and fishes are exposed to salinities as high as 2.5 times that of
normal seawater or 2500 mOsm ("milliosmoles/liter," a term used to
quantify the total concentration of osmotically active solutes in a
solution). A parallel situation occurs in humans; the mammalian
extracellular fluid is regulated at about 300 mOsm but some
mammalian kidney cells are exposed to concentrations as high as
3000 mOsm. Most marine organisms have permeable tissues that are in
direct contact with their environment. Two examples will illustrate
the physiological problems that this generates. The first is an
osmoconformer, or an organism whose body fluids are the same salt
concentration as the seawater that surrounds them. As the salt
concentration of the water rises, so does the salt concentration of
its body fluids. This causes cellular shrinkage which, if
uncorrected, would ultimately result in cell death. Instead, a
process called volume regulation takes place. The second example is
an osmoregulator, or an organism whose body fluids are maintained
at a fixed concentration regardless
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of the salt concentration of the seawater. A fish or a crab can
maintain a blood salt concentration of 300 mOsm. However, gills and
portions of the gut are bathed in seawater that is 1000 mOsm. These
surfaces are permeable, allowing the entry of salts and the escape
of water. This presents two problems: (1) regulating cell volume
and (2) restoring body fluid concentration through osmoregulatory
mechanisms. In a similar manner, components of both of these
processes function in many human organs and, in particular, in the
mammalian kidney. Osmoregulation at the intra- and extracellular
level is made up of the combined mechanisms of fluid and ion
transport and solute or organic osmolyte regulation. The principles
learned from studying osmoregulation in marine animals has
increased the understanding of how the human kidney maintains the
blood at 300 mOsm and also how some kidney cells tolerate the
osmotic stress generated by the kidney's role in concentrating
urine.
With the exception of the halobacteria, the cells of all
organisms have the same adaptive response to a high salt
environment. They accumulate intracellular organic osmolytes. It
was in the Chinese mitten crab, Eriocheir sinensis, that in
1955, (Duchâteau and Florkin, 1955) intracellular
accumulation of amino acids with increased salinity was first
discovered. Various organisms accumulate solutes intracellularly,
but these compounds fall into only a few chemical categories
(Yancey et al., 1982). They are polyols, methylamines, and amino
acids that help compensate for the high salt environment. This
brings up the obvious question of whether there is some highly
conserved mechanism that organisms have in common, or whether the
similarity in adaptive response is the result of convergent
evolution. The hypothesis that there is a conserved mechanism led
to the discovery of an osmotic response element (ORE) (Ferraris et
al., 1994, 1996) in the flanking region of the aldose reductase
(AR) gene, which is responsible for the adaptive accumulation of a
type of polyol during high salt stress. The ORE has also been found
in other genes and in other species (Ruepp et al., 1996; Takenaka
et al., 1994). Hence studies that started with the Chinese mitten
crab have led to the discovery of the osmoregulatory function of
aldose reductase and subsequently to the finding that inappropriate
expression of the same gene in the eye and nerve causes serious
damage in diabetic patients. Knowledge of the aldose reductase gene
is currently being used to determine the mechanism of inappropriate
genetic expression in diabetes.
There are several other examples where studies of properties of
marine organisms have helped elucidate the mechanisms underlying a
variety of biomedical problems caused by defects in fluid or ion
transport. Several of these studies are described below.
The kidney proximal tubules of Pseudopleuronectes
americanus, the winter flounder, and the urinary bladder of
Cancer irroratus, the common red crab, have been used as models
to examine mechanisms of organic anion transport (David S. Miller,
National Institute of Environmental Health Sciences). Organic ion
transport is the cellular mechanism by which kidney cells transfer
potentially toxic
Page 91
compounds from the blood into the urine. These compounds include
drugs, normal and drug metabolites, as well as environmental
pollutants and their metabolites.
The gulf toadfish, Opsanus beta, has been used for
studies on the regulation of nitrogen metabolism and urea
excretion. Most fish excrete nitrogen waste as ammonia, a toxic
compound that is rapidly diluted in water. However, toadfish have
the capability of switching to secreting urea, a much less toxic
metabolite. Urea excretion increases as a response of the fish to
confinement and crowding (Walsh et al., 1994) and appears to be
regulated by mechanisms similar to thos regulating urea transport
in the mammalian kidney (Wood et al., 1998).
The eye of Squalus acanthias, the spiny dogfish shark, is
used to understand mechanisms of vision and fluid formation, with
relevance for human diseases that affect intraocular pressure (like
glaucoma) and lens opacities.
The rectal gland of Squalus acanthias, the major salt
secreting organ in the shark, has proven to be an ideal model
system; atrial natriuretic peptides isolated from the dogfish
heart, have been shown to control sodium chloride excretion from
the rectal gland in combination with vasopressin (Silva et al.,
1996). Atrial natriuretic peptide and vasopressin also regulate
salt and water excretion by the human kidney. Further, because of
the unusually high density of receptors and channels in the shark
rectal gland, it has provided an excellent system for research on
the regulation of chloride secretion in higher vertebrates,
including humans (Forrest, 1996).
The gills of marine organisms form a key interface between the
blood and the environment. The gills of Carcinus maenas, the
green shore crab have provided a useful model for understanding the
regulation of analogous sodium transporters found in the mammalian
kidney (Towle et al., 1997). The gills of Anguilla rostrata,
the American eel, have provided a model system to study how
restructuring of the plasma membrane allows kidney cell membranes
to adapt when salinity changes (Crockett et al., 1996).
The Toadfish:
Its Role in Unraveling the Neural Control of Balance and
Equilibrium
The maintenance of balance and equilibrium in vertebrates is
controlled by the vestibular apparatus (the anatomical structures
concerned with the vestibular nerve, a somatic sensory branch of
the auditory nerve) and its proper functioning is critical for most
organisms including humans (Kornhuber, 1974). The vestibular system
of toadfish has been used for decades as a model for studying
balance and equilibrium. The toadfish was initially chosen for
study because it has a broad flat head that makes it relatively
easy to study the brain and nerves associated with the vestibular
system, and the fish are easy to obtain and adapt readily to the
laboratory. This vestibular system is composed of fluid-filled
canals lined with small hair cells that sense movement of small
crystals, the otoliths. The
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hairs were first studied in the early 1900s by Cornelia Clapp at
the Marine Biological Laboratory in Woods Hole, MA. When the head
moves, the otoliths move, and the hair cells send this information
to the brain. Vestibular systems developed early in the
evolutionary history of vertebrates and did not change greatly as
new species evolved. Thus the vestibular system of the toadfish is
homologous to the vestibular system in humans and can be used to
better understand the basis for human balance disorders.
Horseshoe Crabs: Their Role in
Understanding
Retinal Function and How Eyes See
Knowledge of human vision has its roots in early 20th century
studies of the compound eye of the horseshoe crab and the
phenomenon of lateral inhibition (Sargent, 1987). The horseshoe
crab eye has approximately 1000 photoreceptors whereas the human
retina has more than 100 million photoreceptors. Photoreceptors
perceive and process visual information about the external
environment and then transfer this information to the brain. Hence,
studies of photoreceptors and retinas are relevant to understanding
the neural basis of behavior. Horseshoe crabs and other marine
organisms are particularly amenable to research on retinas because
the tissue is readily accessible and can be removed from the animal
and studied in the laboratory for long periods of time. The neural
network in the horseshoe crab lateral eye is large, there is a
cell-based model of this network, and the animal's behavior in the
field is well known (Passaglia et al., 1997). Further work on the
horseshoe crab may hold the key for deciphering the neurological
basis for vision.
Aplysia: Its Role in Discovering the
Molecular Basis
of Learning and Memory
The marine snail Aplysia has been a valuable model in
studies of neurobiology and behavior. It has a simple brain, made
up of only 20,000 nerve cells, in contrast to the mammalian brain
that comprises around a trillion cells. Moreover, the
Aplysia nerve cells are large and have characteristic
locations, which allow them to be identified in each individual.
This allows the comparison of a nerve cell in the experimental
animals—or those that have learned a response—with the
functionally identical nerve cell in the untrained control animals.
Initial studies delineated a simple behavior, the gill-withdrawal
reflex, and analyzed its neural circuit (Bailey, C.H. et al., 1996;
Kandel et al., 1986). It turned out that even this very simple
reflex can be modified by three different elementary forms of
learning—habituation, sensitization, and classical
conditioning. These forms have similar counterparts in humans.
Furthermore, each of these forms of learning gives rise to both
short-term memory (lasting minutes) and long-term memory (lasting
days to weeks) depending upon the number of training trials. This
work
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provided the first evidence that learning and memory storage
involve changes in strength of synaptic connections made between
nerve cells of the brain. It also showed that a single synaptic
connection can participate in different learning processes and can,
in fact, be modified in different ways by these different learning
processes (Bailey, C.H. et al., 1996; Kandel et al., 1986). Thus, a
single synapse can serve as a site for more than one type of memory
storage and these synaptic storage sites have remarkable
flexibility.
Subsequent studies elucidated the biochemical mechanisms of
memory storage. The investigators began with a short-term form of
learning, sensitization, and studied its effects on one readily
analyzable component of the gill-withdrawal reflex, namely the
synaptic connections between the sensory and motor neurons. They
discovered that the transient strengthening of the synaptic
connections produced by learning involves an increase in
transmitter release. This increase is maintained for the duration
of the learned response (memory). The biochemical process
underlying memory depends on the recruitment of a major
intracellular signaling pathway, the cAMP and cAMP-dependent
protein kinase pathway. These studies provided the first
biochemical insights into the molecular events for short-term
memory storage and illustrated that maintenance of memory is the
result of the persistence of the signal transmitted by cAMP (Silva
et al., 1998).
The conversion of transient, short-term memory to a
self-sustained, long-term memory was also studied in
Aplysia. The switch from short- to long-term memory begins with
the movement to the cell nucleus of the cAMP-dependent protein
kinase. Here, this signaling kinase coordinates the activation of a
number of genes by turning on CREB-1, an activator of gene
expression, and by turning off CREB-2, a repressor of gene
expression. With the activation of CREB-1, new genes are
transcribed that initiate the growth of new synaptic connections
(Silva et al., 1998).
Current studies on mammals confirm that the same principles
pertain to their learning and memory. Thus, the ground breaking
studies on Aplysia have led directly to a molecular
understanding of learning and memory and provide an avenue to more
effective treatments of cognitive disorders (Bailey, C.H. et al.,
1996; Kandel et al., 1986).
The Squid Giant Axon: Its Role in
Establishing How
Nerve Impulses are Conducted
The discovery of the squid giant axon opened up new avenues of
neurobiological research (Baker, 1984; Hodgkin, 1958). The axon,
which is 0.5 mm in diameter or 1000-fold larger than vertebrate
axons, was identified in 1909 by L.W. Williams, who noted that,
"the very size of the nerve processes has prevented their
discovery, since it is well-nigh impossible to believe that such a
large structure can be a nerve fiber." In 1936, J. Z. Young
dispelled the critics through
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his demonstration of an action potential following stimulation
of the giant axon with a crystal of sodium citrate.
Before 1939, nerve action potentials were measured indirectly by
applying electrodes to the outside of nerves, which gave very
limited information. In 1939 Alan Hodgkin and Andrew Huxley began
experimenting with squid giant axons at Plymouth.2 First, Huxley inserted a needle
directly into a fiber, intending that they should measure the
viscosity of the cytoplasm. When that did not work, Hodgkin and
Huxley inserted a glass electrode into the fiber and directly
measured the electrical change when a nerve impulse passed. The
resting potential was 50 mV negative relative to the surrounding
seawater, as expected, but when the fiber was stimulated, the
internal potential reached 50 mV positive. Hodgkin and Huxley
continued their work on the squid axon, showing that the action
potential was propagated by sequential changes in sodium and
potassium ion conductance in the membrane throughout the length of
the axon. Later investigations showed that nerve conduction is
essentially the same in vertebrate neurons as in squid axons. The
proteins that form the channels responsible for the sodium and
potassium conductance have been cloned and examined in molecular
detail, leading to an understanding on an atomic level of the
mechanism of conduction. Throughout these studies the squid giant
axon remained the preferred system, revealing, for example, the
"gating currents" responsible for the changes in conductance that
occur during the action potential (Keynes, 1983). Because of their
large size, it is relatively easy to insert electrodes of all sorts
directly into the neurons without significant damage and it is
possible to collect cytoplasm for biochemical analysis. This has
led to many other breakthroughs, including pioneering studies in
the regulation of intracellular calcium levels and pH.
These revolutionary studies on nerve conduction made possible by
the use of this marine model form the foundation for current
research in neurophysiology, and the concepts that have emerged are
the basis for diagnosis and treatment of disorders of conduction in
nerves and other excitable tissues such as heart and skeletal
muscle.
Use of Fish as Models for Human
Diseases
Fish offer an alternative to rodents for exploring mechanisms of
environmental carcinogenesis. In particular, George Bailey's
laboratory at Oregon State University has promoted the use of the
rainbow trout as a model for research on compounds that cause
cancer. This fish offers the advantages of low rearing costs, an
ultrasensitive bioassay using abundant embryos whose small size
permits observation of tumor development at minute doses of
carcinogen, sensitivity
2 Alan Lloyd
Hodgkin and Andrew Fielding Huxley won the Nobel Prize for
Physiology or Medicine in 1963 for their work with the giant squid
axon.
Page 95
to many classes of carcinogens, well-described tumor pathology,
and responsiveness to tumor promoters and inhibitors (Bailey, G.S.
et al., 1996).
The bicolor damselfish, Pomacentrus partitus, is the
first animal model for one type of human cancer that attacks the
nervous system, neurofibromatosis type 1. This cancer is found in
natural populations of damselfish and has been demonstrated to be
transmissible by injection of extracts of cultured tumor cells.
This observation led to the isolation of a retrovirus, possibly the
causative agent for damselfish neurofibromatosis (Schmale et al.,
1996).
Conclusions
Although some of the most familiar marine animal models were
developed early in this century, the promise of using these
organisms in research is rediscovered roughly every decade (NIEHS
News, EHP 102:272). The discovery of marine organisms as useful
models for biomedical research has frequently been serendipitous,
but many successful marine models have emerged from a thorough
understanding of the natural history and basic biology of marine
organisms. Strategies for supporting biomedical research through
the use of marine models include:
•
Encourage education and research in natural history, taxonomy,
physiology, and biochemistry of marine organisms as the foundation
for the development of valuable new models for biomedical research.
As science, and biology in particular, grows more and more
compartmentalized into sub-disciplines, it becomes increasing
important to foster interdisciplinary approaches to biomedical
problems through educational and research opportunities.
•
Promote the development of nonmammalian models for biomedical
research and laboratory culture of targeted marine species. The
Comparative Medicine program at the National Center for Research
Resources at the NIH currently supports research to explore and
develop alternative animal models for biomedical research and funds
centers that supply laboratory reared Aplysia and various
cephalopods (squid, cuttlefish, and octopus). This type of program
is important because it provides researchers with a dependable
source of experimental animals that can be bred to both reduce
individual variability and to develop new genetic strains. Also,
collection of marine animals from natural populations in some cases
risks the depletion of the species in the wild. Development of
culture techniques and facilities for marine organisms will give
more alternatives to the use of mammals in research and will
promote our understanding of complex biomedical problems.
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