TOXICOLOGIC PATHOLOGY ISSN:0192-6233 Volume 18, Number I (Part I), 1990

Copyright 1990 by the Society of Toxicologic Pathologists Printed in U.S.A.

Acute Neurotoxicity of Domoic Acid in the Rat*

LEANDER TRYPHONAS, JOHN TRUELOVE, EDWARDO NERA, AND FRANK IVERSON
Toxicology Research Division, Bureau of Chemical Safety, Food Directorate,
Health Protection Branch, Health and Welfare Canada, Ottawa, KiA OL2

ABSTRACT

A recent outbreak of human food poisoning, characterized by severe gastrointestinal and neurologic ab-
normalities, with a fatal outcome in 3 patients, was attributed to the consumption of poisonous mussels
containing domoic acid at an abnormally high concentration. The purpose of the present study was to
determine if domoic acid, a glutamate analogue extracted from poisonous mussel, was neurotoxic to rats.
Groups offemale Sprague-Dawley rats were dosed once intraperitoneally with 0, 1, 2, 4, or 7.5 mg domoic
acid/kg of body weight and observed for a maximum period of 24 hr. Clinically, control rats and rats in the
I mg/kg group were unremarkable. Seventy-five percent of the animals in the 2 mg/kg group had equivocal
transient behavioral signs. One that was given 2 mg/kg and all rats given in excess of 4 mg/kg of body weight
developed unequivocal behavioral and neurologic signs culminating in partial seizures and status epilepticus.
Histopathologically, severely affected rats developed selective encephalopathy characterized by neuronal
degeneration and vacuolation of the neuropil in the limbic and the olfactory systems, and retinopathy
characterized by neuronal hydropic degeneration of the inner nuclear layer and vacuolation of the external
plexiform layer. The results of this study suggest that domoic acid is excitotoxic and causes a characteristic
syndrome with clinical signs and histopathologic lesions similar to those reported for kainic acid.
Keywords. Dendrotoxic lesions; excitotoxins; gliotoxic lesions; hippocampus; neurotoxins; rodent; toxic
encephalopathy; toxic mussels

INTRODUCTION citation when applied iontophoretically and neu-

In the last quarter of 1987, approximately 175
Canadians became moderately to seriously ill after
consuming freshly harvested cultivated blue mus-
sels (Mytilus edulis L.) originating in eastern Prince
Edward Island. Clinically, early symptoms included
nausea, vomiting, and disorientation. Irreversible
neurologic signs developed in 3 persons who even-
tually died 2-3 weeks later.
The agent allegedly responsible for this outbreak
was domoic acid, known to be produced by the red
alga Chondria armata. The diatom Nitzschia pun-
gens, found in waters where mussels are cultivated,
is now suspected to be the source of domoic acid in
this instance (6, 10, 15,20,27). Mussels associated
with the recent outbreak of marine food poisoning
were found to contain domoic acid in excess of 800
ug/g (11, 15,27). Under normal conditions, domoic
acid has not been previously detected in mussels.
Domoic acid is structurally related to kainic acid,
which is a rigid analogue ofthe putative neurotrans-
mitter L-glutamate and which causes neuronal ex-
ronal death when higher concentrations are applied
(4, 25, 26). The anions of domoic acid (and quis-
qualic acid) were found to be at least 2 orders of
magnitude more potent than L-glutamate, and equal
to or stronger than kainate, when tested on rat spinal
motoneurons by microelectrophoresis and on frog
spinal motoneurons by superfusion of the procaine-
blocked hemisected spinal cord in vitro (2, 3). Kainic
acid has been extensively used as a means for se-
lectively lesioning neuronal cell bodies while sparing
axons en passant (5).
To determine the biologic effects and the dose
response curve of domoic acid present in contam-
inated toxic mussels, a number ofexperiments were
carried out in our laboratories using mice, rats, and
monkeys (Cynomolgus fascicularis) (10). The pur-
pose of the present report is to document the toxicity
of domoic acid in the rat using clinical aberrations
and histopathologic lesions as indices of neurotox-
icity.
MATERIALS AND METHODS

* Address correspondence to: Dr. Leander Tryphonas, Pa- Animals

thology Section, SirFrederick G. Banting Research Centre, Health
Protection Branch, Tunney's Pasture, Ottawa, Ontario, Canada Seventeen female Sprague-Dawley rats weighing
KIA OU. 180 15 g were obtained from Charles River, S1.

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2 TRYPHONAS ET AL
TABLE I.-Summary of study design.
Observation
Dose Volume period
(mg/kg) (ml) No. of rats
1.0 2 4
2.0 2 4
4.0 2 4
7.5 2 1
0.0 2 4
Constant, Quebec. They were housed in group banks
in a humidity-, light-, and temperature-controlled
room, and maintained on laboratory chow (Purina
Rodent Laboratory Chow (R) #5001, St. Louis, MO).
Feed and water were provided ad libitum.
Chemical
Crude domoic acid (85%) used for the intraperi-
toneal injections was isolated from toxic mussel ex-
tracts by the Food Research Division ofHealth Pro-
tection Branch, Health and Welfare Canada, using
repetitive preparative HPLC. Mussel extracts, pre-
pared according to the method suggested for the
bioassay of paralytic shellfish poison (l0) and con-
taining elevated levels of domoic acid, were used as
the starting material.
Dosage
Rats were placed individually in clear plastic bins
and dosed once intraperitoneally with 0, 1, 2, 4, or
7.5 mg domoic acid/kg of body weight dissolved in
physiologic saline. The dose volume was adjusted
with physiologic saline so that each rat received 2
ml (Table I).
Clinical Evaluation
Immediately after the administration of domoic
acid, the rats were replaced in the clear plastic bins
and observed continuously for the first 4 hr and
intermittently for a maximum period of 24 hr. An-
imals that developed severe clinical signs were killed
earlier (Table I). Clinical signs were recorded as they
appeared. Complex clinical signs were grouped into
minor seizures, status epilepticus, and crab-like
walking- based on criteria established previously
(19) and on our own observations. A minor seizure
was considered to take place when 1 or more of the
following signs occurred intermittently: forelimb
tremor, head nodding, circling, loss of postural con-
trol, mastication and profuse salivation, praying and
rearing, scratching, and wet dog shakes. Status epi-
lepticus included continuous occurrence of agita-
tion, forelimb tremor, generalized tremor, praying
and rearing with falling, and profuse salivation. Crab-
like walking was defined as the combination of se-
vere kyphosis, tucked-up abdominal musculature,
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TOXICOLOGIC PATHOLOGY
(hr)
24
6-24
4-6
2
24
FIG. 1.- Rat treated with 4 mg domoic acid/kg ip. Emp-
ty mastication, salivation, and paw clasping were early
signs of impending seizure.
increased tone oflimb muscles, and raising the body
off the ground when walking.
Morphologic Evaluation
All rats were killed under deep chloroform an-
esthesia by intracardiac perfusion with heparinized
lactated Ringer's solution, followed by 200 ml of
fixative consisting of 2% glutaraldehyde and 1%
paraformaldehyde in 0.1 M sodium cacodylate buff-
er, pH 7.2.
A complete necropsy was performed and the fol-
lowing tissues were immersed in 10% neutral buff-
ered formalin for paraffin histologic processing: eyes,
optic nerves, olfactory bulbs, brain, and spinal cord
with dorsal root ganglia. The brain and the thoracic
and lumbar enlargements of the spinal cord were
sliced into blocks of approximately 2 mm thickness
and embedded in paraffin. Sections were cut at ap-
proximately 6 and 10 11m thickness and stained with
hematoxylin and eosin, luxol fast blue, Bodian, and
Nissl methods. Selected sections were stained with
the PAS and Holzer's methods. Selected semithin
sections were stained with toluidin blue.
Total interneuronal vacuolar area in the 5 affected
and 5 control rats was estimated morphometrically
in the hippocampus using a Zeiss lEAS 2 image
analysis system interfaced to a Hamamatsu video
camera on a Zeiss Photomicroscope III. Areas were
measured from CA3a and CA3b fields of the rostral
and caudal regions of the hippocampus.
RESULTS
Clinical Findings
Control rats and rats in the 1 mg/kg group were
unaffected. Three rats in the 2 mg/kg group devel-
oped transient clinical signs suggestive of central
nervous system disturbance. Soon after the intra-
peritoneal injection there was a period of inertia
during which the rats remained motionless. Later,
they developed moderate exploratory behavior, kept
Vol. 18, No.1 (Part 1), 1990 DOMOIC ACID TOXICITY IN THE RAT 3

in apparently purposeless exploratory behavior, rats

FIG. 2.- Rat treated with 4 mg domoic acid/kg ip. Lum-
bar kyphosis, raised body (crab-like walking), and partial
enophthalmos with squinting appeared intermittently prior
to seizure.
their ear lobes pulled back, had occasional unilateral
scratching of the scapular region by the ipsilateral
hind paw, and remained withdrawn for varying pe-
riods oftime but were fully recovered within 24 hr.
In addition to becoming withdrawn and engaging
in the 2 (one rat), 4, and 7.5 mg/kg groups developed
hunched posture and piloerection of the vibrissae,
reoccuring enophthalmos, occasional wet dog shakes,
waddling gait, and bilateral scratching of an area of
the skin extending from the caudal border of the
scapula to the lateral canthus of the eye by the ip-
silateral hind paw. Further, head nodding, protract-
ed chewing and salivation, rearing and/or "praying"
with forelimb extension and forepaw clasping or
clapping, generalized fine tremor, rigidity of move-
ment giving rise to a "crab-like" walking, loss of
postural control ending in rolling followed by sud-
den regaining of posture, circling, and occasional
jumping were superimposed clinical signs that ap-
peared later (Figs. 1 and 2). Rats in a more advanced
stage of disease also leaned against objects, sat on
their hind limbs and tail, and developed intermit-
tent status epilepticus, gasping, and cyanosis fol-
lowed by intervening periods of flaccidity (Fig. 3).
At this point, severely affected animals were eu-
thanatized. The specific type and the number of
signs seen were dose-dependent (Fig. 4).

FIG. 3.-Stages in the evolution of a typical seizure in a rat treated with 4 mg domoic acid/kg ip: A) Pulling back of
the ear lobes, roughened coat, and near normal posture; B) Praying (anterior part of body raised with the head extended,
mouth open, ear lobes pulled back, forelimbs extended, and front paws clasped); C) Sitting on hind legs and tail with
front paws tightly clasped; and D) Loss of balance with early temporary falling.
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4 TRYPHONAS ET AL TOXICOLOGIC PATHOLOGY

1mg Domoic/kg
CHEWING
DEATH
EXPLORING
HEAD JERKS
KYPHOSIS
REARING
SCRATCHING
SEIZURES
WIDE STANCE
WITHDRAWN

20 40
2 mg Domoic/kg
CHEWING
DEATH
EXPLORING
HEAD JERKS
KYPHOSIS
REARING
SCRATCHING
SEIZURES

WIDE STANCE
WITHDRAWN

20 40 60 80 100 120
Time
4mg Domoic/kg
CHEWING
DEATH


EXPLORING
HEAD JERKS


KYPHOSIS
REARING

SCRATCHING
SEIZURES


WIDE STANCE

WITHDRAWN

20 40 60 80 100 120
Time
7.5 mg Domoic/kg
CHEWING
DEATH

EXPLORING

HEAD JERKS
KYPHOSIS


REARING
SCRATCHING

SEIZURES
WIDE STANCE
WITHDRAWN

20 40 60 80 100 120 140

Time
FIG. 4.-Chronologic appearance and evolution ofcJinical signs in treated S-D rats: The time to onset for most cardinal
signs was inversely proportional to dose size. Control rats were unremarkable.

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Vol. 18, No.1 (Part 1), 1990 DOMOIC ACID TOXICITY IN THE RAT 5

TABLE n.-Distribution and frequency of cerebral and retinal domoic-acid-induced damage in Sprague-Dawley rats.
Domoic acid (mg/kg bodyweight)
4
2 7.5 0
Lesioned area (230) (48) (228) (229) (217) (47) (C)

Cerebrum
Amygdaloid complex
Bed. n. stria terminalis +
Medial amygdaloid n. + + +
Posterolat. cort. amyg. n. +
Posteromed. cort. amyg. n. +
Cortex
Cingulum + + + +
Entorrhinal cortex +
Frontoparietal cortex + +
Retrospl. Str. 18 cortex +
Hippocampus
CAl field + + + +
CA3 field + + + + + +
CA4 field + +
Subiculum + +
Tenia tecta (ant. hippoc.) +
Hypothalamus
Arcuate nucleus + + + + +
Lateral hypothalamic area +
Septohypothalamic n. +
Suprachiasmatic n. +
Olfactory system
Anterior olfactory n. + + +
Bed n. access. olf. tract +
Endopyriform n. + + +
Olf. bulb, ext. plex. layer +
Primary olfactory cortex + + + + +
Septum
Lat. septal n. ventralis + +
Eye
Retina
Inner nuclear area + + + +
Outer plexiform layer + + +
( ) rat identification number.

Gross Findings A common histologic finding in the brain of af-

Rats that developed advanced clinical signs had
unkempt fur and wet hair on the front limbs, around
the mouth, over the ventral aspect of the neck and
abdomen, and at the perineal region due to excessive
salivation and incontinence. No lesions were seen
in treated rats that failed to develop clinical signs.
Control rats were unremarkable.
Histopathologic Findings
Control rats, as well as rats treated with 1 mg
domoic acid/kg body weight, and 3 of 4 rats given
2 mg/kg were unremarkable. One rat given 2 mg/
kg and rats in the 2 higher treatment groups devel-
oped acute selective encephalopathy and retinopa-
thy. The spinal cord was unaffected. The regional
distribution and frequency of tissue damage are
shown in Table II.
fected rats included: vacuolation in the neuropil,
neuronal hyperchromasia and shrinkage with mul-
tiple indentations of the somatic profile, and astro-
cytic swelling (Fig. 5). More advanced lesions in-
cluded neuronal necrosis (Fig. 6).
The distribution pattern oflesions in cerebral nu-
clei was noticeably different from that seen in struc-
tures possessing a cortical organization. In the for-
mer, lesions were diffuse and consisted of
pronounced vacuolation of the neuropil, moderate
astrocytic swelling, and occasional neuronal hyper-
chromasia and shrinkage. In the latter, the lesions
had a laminar distribution. Thus, lesions ofthe pri-
mary olfactory cortex were prevalent in layers II and
III and only occasionally extended to deeper layers.
Lesions of the frontoparietal cortex were confined
to the three deepest layers. Their presence was be-
trayed by a diminished staining of the affected area.

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6 TRYPHONAS ET AL
FIG. 5.-Typical domoic-acid-induced CNS changes 4
hr post-treatment. Pronounced vacuolation of the neu-
ropil, neuronal shrinkage, multiple indentations of so-
matic profiles, and astrocytic swelling in the stratum py-
ramidale of hippocampus field CA3. H&E. x 600.
Lesions in the hippocampus were peculiar in that
they were limited to the pyramidal cell layer ofCA3
and, less often, CAI and CA4 zones, the outer region
of the stratum oriens, and the stratum lacunosum
moleculare. Dense microvacuolation and occasion-
al swollen glial cells were the only lesions seen in
the stratum oriens near the alveus (Fig. 7A) and in
the stratum lacunosum moleculare (Fig. 7B). In the
pyramidal cell layer, the neurons and the proximal
end of their dendrites were darkened, had angular
or indented profiles, and were surrounded by vari-
ably sized vacuoles which at times were also seen
in the stratum lucidum (Fig. 7C). Amongst the dis-
tended vacuolar profiles, and occasionally just out-
side the pyramidal cell layer proper, there were swol-
len astrocytes with hydropic cytoplasm and/or
nucleus. Hydropic astrocytic nuclei often were only
represented by an ill-defined nuclear envelop.
Unaided light microscopic examination revealed
that the severity of hippocampal lesions increased
in a rostrocaudal direction. Morphometric analysis
offield CA3 changes demonstrated that vacuolation
of the neuropil was almost twice as extensive in the
caudal part than in the rostral end ofthe hippocam-
pus. Changes in field CA3 of the hippocampus and
in layers II and III of the primary olfactory cortex
were present in all rats that developed unequivocal
clinical signs of domoic acid toxicity (Table 11).
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TOXICOLOGIC PATHOLOGY
FIG. 6.-Moderate vacuolation of the neuropil and neu-
ronal necrosis in the arcuate nucleus of a domoic-acid-
treated S-D rat (7.5 mg/kg). H&E. x 100.
Ocular lesions were most often found in the outer
two-thirds of the inner nuclear layer and the inner
half of the external plexiform layer (Table II). In the
inner nuclear layer, affected bipolar and horizontal
cells had undergone hydropic degeneration and/or
necrosis (Fig. 8). Typically affected cells had mas-
sively enlarged cytoplasm which either was vacuous
and edematous or contained a scant amount of ve-
siculated cellular debris. In addition to being en-
larged, the nuclei had margination of chromatin,
were karyorrhectic, or had undergone pyknosis. In
some instances, pyknotic nuclei were surrounded by
a thin clear halo which in tum was bound by a thin
membrane. In the outer plexiform layer, there was
moderate microvacuolation which most often oc-
curred along the inner halfofits width. The vacuoles
were round, occurred singly or in clusters, and only
rarely contained a small amount of cellular debris.
DISCUSSION
The results of this study indicate that a single
intraperitoneal administration ofdomoic acid to rats
at doses exceeding 2 mg/kg of body weight produces
a well defined neurologic syndrome characterized
by scratching, "wet dog shakes," empty mastica-
tions, salivation, generalized fine tremor, seizures,
and status epilepticus in that order. Its morphologic
correlates include selective encephalopathy and ret-
inopathy. Both the neurologic manifestations and
the neuropathologic lesions closely resemble those
reported for kainic acid, which is a well studied
excitatory amino acid structurally related to domoic
acid and L-glutamate and which has a specific type
of selectivity for CNS damage (13).
Vol. 18, No. 1 (Part 1), 1990 DOMOIC ACID TOXICITY IN THE RAT
FIG. 7.-Hippocampus ofa treated S-D rat (4 mg/kg).
A) Dilated profiles in the stratum pyramidale and dense
microvacuolation and occasional swollen glial cells in the
outer zone of the stratum oriens in field CA I; B) Dense
dilated profiles in the stratum lacunosum moleculare ; and
C) Dilat ed profiles are prominent in the strata pyramidal e
and lucidum of field CA3. H&E. x 250.
Despite the close qualitative similarity with kai-
nic-acid-induced lesions reported in the literature,
there were notable differences in the distribution of
target sites in the brain and the eyes between do-
moic-acid- and kainic-acid-treated rats. In the brain,
kainic acid is known to damage more areas of the
cerebral cortex and more nuclei in the olfactory and
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7
FIG. 8.-Retina of treated SoD rat (7.5 mg/kg). Mod-
erate hydropic degeneration and nuclear pyknosis in cells
of the inner nuclear layer and variably sized dilated pro-
files in the external plexiform layer. Toluidine blue. x 250.
limbic systems than was the case with domoic acid
(19). In the retina, kainic acid has a predilection for
the inner plexiform and the inner half of the inner
nuclear layers (13, 23). In contrast, domoic acid
caused severe edema and necrosis of the somata and
processes ofcells in the outer two-thirds of the inner
nuclear layer and vacuolation in the external plex-
iform layer. The reason for such differences is not
readily apparent, as neither the operant neurotrans-
mitter nor the distribution of domoic acid receptors
in the retina have been determined. However, do-
moic-acid-induced lesions seem to occur at the first
level of synapses of the visual pathway. The occur-
rence oflesions at this predominantly glutamatergic
site lends support to Olney's view that development
of lesions is mediated by glutamate (9, 16, 17).
In addition, the extent of damage reported in tar-
get sites common to both compounds was greater
in animals treated with kainic acid (17-19,24). Fur-
thermore, it was reported that kainic-acid-induced
lesions in the olfactory cortex and parts of the amyg-
daloid complex become more severe with the pas-
sage of time after treatment (19, 24). These differ-
ences are difficult to reconcile at this time especially
because domoic acid is known to be at least 2 orders
of magnitude more potent than L-glutamate and
equal to or stronger than kainate (I , 8). It is not clear
whether these differences are due to drug specificity,
to drug potency, or to the relatively shorter exposure
time allowed domoic acid animals in the present
experiment. Present scarcity ofdomoic acid has pre-
vented us from studying the effects ofa wider dosage
range. However, given the proven higher potency
of domoic acid and until further mechanistic ex-
periments are carried out , it is reasonable to assume
that our rather short post-treatment period is the
most likely reason for the apparent discrepancy.
The exact mechanism by which domoic acid ex-
8 TRYPHONAS ET AL
erts its deleterious effecton the neuron is not known.
However, given the similarity with kainic-acid-in-
duced lesions, the close structural resemblance to
kainic acid, and the previously published results on
kainic acid toxicity (13, 19), it would be reasonable
to suggest that the operant mechanism by which
domoic acid is causing cerebral and ocular damage
is like that of kainic acid. Originally, kainic acid
toxicity was thought to be mediated by an excess of
synaptic glutamate. Presently, action of kainic acid
is hypothesized to be at a site on the synaptic mem-
brane which is distinct from the post-synaptic glu-
tamate receptor. Binding of kainic acid to the post-
synaptic membrane is believed to potentiate greatly
the excitatory action of the glutamate and at the
same time inhibit the uptake of glutamate by the
nerve ending or nearby glia. This, in turn, further
potentiates kainic acid action in the synaptic cleft
(12, 21). Although the structural and biologic sim-
ilarities between domoic and kainic acids suggest
that domoic acid toxicity also is mediated by glu-
tamate, there is little direct evidence in the literature
to indicate that domoic acid binds to the postsyn-
aptic membrane. Meldrum classifies domoic acid
with kainate agonists, suggesting that domoic acid
binds to kainic acid receptors (14).
Whereas kainic-acid-induced neuronal degener-
ation and neuropilic vacuolation have been exten-
sivelystudied, the pathogenesisof gliallesions, which
were also present in the domoic-treated rats, has
received little attention. Neuronal damage and most
neuropilic vacuolation are considered to be due to
acute energy depletion resulting from continuous
cellular excitation. In contrast, the mechanism of
acute glial hydropic degeneration remains unde-
fined. It has been suggested, however, that binding
of kainic acid to a subclass of glutamate receptors
stimulates seizure-related sustained excitation. The
immediate, possibly NA +-related, response is seen
as astrocytic or dendritic swelling (7, 17, 19). Also,
there is growing evidence that glial cells participate
in the metabolism of glutamate by removing un-
coupled glutamate from the synaptic cleft, convert-
ing it to glycine, and returning it to the presynaptic
axon (22). It is, therefore, tempting to speculate that
glialdegeneration at the site ofselective domoic acid
damage may prolong the excitatory activity of glu-
tamate by preventing glutamate uptake by lesioned
resident astrocytes.
The results of the present study indicate that, in
rats, the threshold dose ofdomoic acid administered
intraperitoneally lies between I and 2 mg/kg ofbody
weight. The close resemblance of domoic-acid-in-
duced toxicity with that ofkainic acid suggests that
they share a common pathogenesis. Additional
studies are needed mainly to determine whether dif-
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TOXICOWGIC PATHOLOGY
ferences in target site distribution and severity of
damage between domoic acid and kainic acid tox-
icity are compound specific or are due to differences
in dosage and length ofexposure. It is also necessary
to establish whether domoic acid utilizes kainic acid
post-synaptic receptors or makes use of distinct re-
ceptors. Finally, it is important to ascertain the ex-
tent of eNS damage produced by extracts of toxic
mussels containing domoic acid.
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