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The Journal of Neuroscience, December 15, 1999, 19(24):10985-10992
Norepinephrine-Deficient Mice Have Increased Susceptibility to
Seizure-Inducing Stimuli
Patricia
Szot1, 6,
David
Weinshenker2, 5,
Sylvia S.
White1,
Carol A.
Robbins3,
Nicole C.
Rust2, 5,
Philip A.
Schwartzkroin3, 4, and
Richard D.
Palmiter2, 5
Departments of 1 Psychiatry and Behavioral Science,
2 Biochemistry, 3 Neurological Surgery, and
4 Physiology/Biophysics and 5 Howard Hughes
Medical Institute, University of Washington, Seattle, Washington 98195, and 6 GRECC, Puget Sound Health Care System,
Seattle, Washington 98108
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ABSTRACT |
Several lines of evidence suggest that norepinephrine (NE) can
modulate seizure activity. However, the experimental methods used in
the past cannot exclude the possible role of other neurotransmitters coreleased with NE from noradrenergic terminals. We have assessed the
seizure susceptibility of genetically engineered mice that lack NE.
Seizure susceptibility was determined in the dopamine  -hydroxylase
null mutant (Dbh  /) mouse using four different convulsant stimuli: 2,2,2-trifluroethyl ether (flurothyl),
pentylenetetrazol (PTZ), kainic acid, and high-decibel sound.
Dbh / mice demonstrated enhanced susceptibility
(i.e., lower threshold) compared with littermate heterozygous
(Dbh +/) controls to flurothyl, PTZ, kainic acid, and
audiogenic seizures and enhanced sensitivity (i.e., seizure severity
and mortality) to flurothyl, PTZ, and kainic acid. c-Fos mRNA
expression in the cortex, hippocampus (CA1 and CA3), and amygdala was
increased in Dbh / mice in association with
flurothyl-induced seizures. Enhanced seizure susceptibility to
flurothyl and increased seizure-induced c-fos mRNA expression were
reversed by pretreatment with
L-threo-3,4-dihydroxyphenylserine, which partially restores
the NE content in Dbh / mice. These genetically
engineered mice confirm unambiguously the potent effects of the
noradrenergic system in modulating epileptogenicity and illustrate the
unique opportunity offered by Dbh / mice for elucidating the pathways through which NE can regulate seizure activity.
Key words:
dopamine -hydroxylase; c-fos mRNA; norepinephrine; flurothyl; epilepsy; seizure; kainic acid
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INTRODUCTION |
Chen et al. (1954) first suggested
that the noradrenergic system modifies seizure activity. Since then,
four major observations have supported an anticonvulsant role for
norepinephrine (NE): (1) selective lesioning of noradrenergic neurons
(with 6-hydroxydopamine or DSP-4) increases seizure susceptibility to a
variety of convulsant stimuli (Arnold et al., 1973; Jerlicz et al.,
1978; Mason and Corcoran, 1979; Snead, 1987; Trottier et al., 1988;
Sullivan and Osorio, 1991; Mishra et al., 1994); (2) direct stimulation
of the locus coeruleus (LC, the major concentration of noradrenergic cell bodies in the CNS) and the subsequent release of NE reduce CNS sensitivity to convulsant stimuli (Libet et al., 1977; Turski et
al., 1989); (3) genetically epilepsy-prone rats (GEPRs), a widely used
animal model of epilepsy, have deficient presynaptic NE content, NE
turnover, tyrosine hydroxylase levels, dopamine -hydroxylase (DBH)
levels, and NE uptake (Jobe et al., 1984; Dailey and Jobe, 1986;
Browning et al., 1989; Lauterborn and Ribak, 1989; Dailey et al.,
1991); and (4) adrenergic agonists acting at the  -2 adrenoreceptor
(2-AR) have anticonvulsant action (Papanicolaou et al., 1982; Baran
et al., 1985; Loscher and Czuczwar, 1987; Fletcher and Forster, 1988;
Jackson et al., 1991).
Although there is significant evidence that the NE system is
anticonvulsant, there are several considerations that temper one's
confidence in the hypothesis that NE, itself, reduces seizure sensitivity. For example, although the lesioning studies (i.e., chemical destruction of noradrenergic terminals) reduce the amount of
NE release, this manipulation also reduces the release of other transmitters coreleased with NE. The neuropeptides galanin and neuropeptide Y (NPY) and the neurotransmitter adenosine (i.e., ATP) are
released at noradrenergic terminals and have been shown to exert
anticonvulsant effects against several convulsant stimuli (Murray et
al., 1985; Mazarati et al., 1992, 1998; Dichter, 1994; Erickson et al.,
1996; Baraban et al., 1997). A similar argument can be made for the
anticonvulsant effect of direct LC stimulation, which results in the
release not only of NE but also of these cotransmitters. The enhanced
seizure sensitivity of the GEPRs may not be caused solely by their
abnormal noradrenergic system, because these animals also have
abnormalities in their central serotonergic, GABAergic, and excitatory
amino acid systems (Faingold et al., 1986; Dailey et al., 1992;
Meyerhoff et al., 1992); moreover, other animal models of epilepsy have
a higher than normal central NE content (Noebels, 1986; Hara et al.,
1993). Finally, the 2-AR pharmacological studies are difficult to
interpret because the effect of clonidine (2-AR agonist) on
seizure-induced activity can be biphasic, nonexistent, or even
proconvulsant (King and Burnham, 1982; Tacke and Kolonen, 1984;
Lapin and Ryzor, 1990). Such multiple responses to 2-AR agonists may
be caused by the localization of the affected 2-AR. Activation of
presynaptic 2-AR autoreceptors would reduce transmitter released at
NE terminals (L'Heureux et al., 1986), whereas activation of
postsynaptic 2-ARs would mimic the effect of released NE. Because it
has not been determined whether the anticonvulsant effect of 2-AR
agonists is mediated via pre- or postsynaptic receptors, it remains
unclear whether increased NE release is anti- or proconvulsant.
Taken together, these studies suggest that changes in noradrenergic
functions (terminal NE content or release) can modulate seizure
activity, but they do not resolve the issue of whether NE is, itself,
anticonvulsant. It is this issue that we have addressed with the DBH
null mutant (Dbh /) mouse. These animals selectively lack NE and epinephrine (dopamine content tends to be elevated) because
DBH is required for the conversion of dopamine to NE (Thomas et al.,
1998).
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MATERIALS AND METHODS |
Animals. Mice were derived from a hybrid line
(129/Sv/Ev and C57BL/6J). Dbh / and heterozygote
(Dbh +/) mice were bred as described previously (Thomas et
al., 1995). Mice were maintained on a 12 hr light/dark cycle in a
specific pathogen-free facility at the University of Washington
(Seattle, WA). Food and water were available ad libitum, and
animals were maintained according to the guidelines outlined in the
NIH Guide for Care and Use of Laboratory Animals. All animal
procedures were approved by the University of Washington Animal Care
Committee. Genotype was deduced from phenotype (Dbh /
mice exhibit delayed growth during adolescence and ptosis), and a
subset of mice was confirmed by PCR (Thomas et al., 1995).
Dbh +/ mice are indistinguishable from wild-type (+/+)
mice as to NE and epinephrine levels (Thomas et al., 1998). Preliminary
studies showed no significant difference in seizure susceptibility
[2,2,2-trifluroethyl ether (flurothyl)-induced seizures] between
wild-type (+/+) and heterozygote Dbh (+/) mice; therefore,
Dbh +/ mice were used as controls in all experiments. Adult (3-6 months) male and female littermates of each genotype were
evenly distributed to experimental and control groups for each
convulsant stimulus. A subset of animals will receive a single intraperitoneal injection of
L-threo-3,4-dihydroxphenylserine (DOPS; 1 mg/gm). DOPS is converted to NE by aromatic
L-amino acid decarboxylase, which is present in
all biogenic amine neurons. Five hours after a single administration of
DOPS, NE levels peak in peripheral and central regions; dopamine levels
are not affected by DOPS (Thomas et al., 1998).
Flurothyl susceptibility. Flurothyl seizure thresholds were
determined for Dbh +/ and Dbh / mice, with
and without previous administration of DOPS. Mice were placed in an
air-tight Plexiglas chamber, and the volatile convulsant
flurothyl (Aldrich, Milwaukee, WI) was infused (20 µl/min)
onto filter paper from which it vaporized (Prichard et al., 1969). The
latencies (seconds) to the first myoclonic jerk (focal seizure) and to
generalized (clonic/tonic) seizure served as the measurements of
seizure susceptibility. Each mouse was tested individually, removed
immediately from the chamber after seizure onset, and received only one
exposure to flurothyl. Some animals received DOPS (1 mg/gm, i.p.) 6 hr
before seizure-threshold testing. Latency (seconds) data from each
group were expressed as the mean ± SEM and were analyzed
with Student's t test comparisons; statistical significance
was taken at p < 0.05. For each group (Dbh
/ and Dbh +/ mice, with and without DOPS), we also
determined the percentage of animals proceeding to tonic extension
followed by recovery versus the percentage progressing to death.
Surviving animals were killed 1 hr after the seizure to measure c-fos
mRNA expression.
Pentylenetetrazol susceptibility. Pentylenetetrazol (PTZ) at
two different doses (30 and 40 mg/kg, i.p.) was administered to both
Dbh +/ and Dbh / mice. After injection, the
animals were placed into a clear container and closely monitored for 10 min. The latencies (seconds) to the first myoclonic jerk (focal seizure), to forelimb clonus, and to generalized (clonic/tonic) seizures were measured and analyzed as described above.
Kainic acid susceptibility. Kainic acid (stock solution, 4 mg/ml) was dissolved in neutral-buffered saline and administered to
both Dbh +/ and Dbh / mice at an
intraperitoneal dose of 20 mg/kg. After injection, the animals
were placed into a clear container and closely monitored for 40 min.
The latency (seconds) to the first generalized (clonic/tonic) seizure
was measured and was analyzed as described above. For each group, we
also determined the percentage of animals that progressed to death.
Audiogenic seizure susceptibility. Audiogenic seizure
sensitivity in Dbh +/ and Dbh / mice was
determined by exposing animals to a 115 dB sound for 60 sec with an
SR Pilot (San Diego Instruments, San Diego, CA). After the sound
was started, the mouse was closely monitored for occurrence of a
seizure. If no seizure occurred, the sound was terminated after 60 sec.
If a seizure was observed during the 60 sec period, the sound was
immediately terminated, and the animal was removed. Mice were scored as
exhibiting or not exhibiting a seizure.
c-Fos mRNA expression after flurothyl-induced seizures. The
mice that survived flurothyl-induced generalized seizures were killed
by cervical dislocation 1 hr after the seizure [Dbh +/ (n = 8) and Dbh / (n = 6) without DOPS; Dbh +/ (n = 8) and Dbh / (n = 9) with DOPS]. To determine
basal c-fos mRNA expression Dbh +/ (n = 6)
and Dbh / (n = 6) mice (same age as the
flurothyl-tested mice) were also killed. Brains were collected from
each animal and immediately frozen on dry ice. Twenty micrometer
coronal sections containing neocortex and hippocampus were cut on a
cryostat and mounted onto Fisher Superfrost slides (Fisher Scientific,
Houston, TX). Slides were stored at 70°C until assayed.
Tissue preparation and labeling of the c-fos oligonucleotide was
performed as described previously (Szot et al., 1997). The c-fos
oligonucleotide probe was a 51-base probe complementary to nucleotides
270-319 of the c-fos mRNA (Curran et al., 1987). The oligonucleotide
probe was 3'-end-labeled with [33P]dATP
(New England Nuclear, Boston, MA) using terminal deoxyribonucleotidyl transferase (Life Technologies, Gaithersburg, MD) and then purified on
NEN-Sorb columns (New England Nuclear). The c-fos hybridization buffer
for the flurothyl-induced seizure assay contained 0.3 × 106 cpm/50 ml. The c-fos hybridization
buffer for the basal assay contained 0.4 × 106 cpm/50 ml. Hyperfilm (Amersham,
Arlington Heights, IL) was exposed to slides containing tissue
hybridized with c-fos
[33P]oligonucleotide for 1 d for
the flurothyl-induced seizure assay and 5 d for the basal assay.
To quantitate c-fos mRNA expression in the specific regions of the CNS,
all sections were processed, hybridized, and washed in the same
experimental session. Each sheet of Hyperfilm contained sections from
all four groups (Dbh +/ and Dbh / mice with
and without DOPS). To determine basal c-fos mRNA expression, sections
from Dbh +/ and Dbh / mice were processed,
hybridized, and washed in a similar manner. Optical densities were
measured from films using the MicroComputer Imaging Device
(Imaging Research, Ontario, Canada). Separate optical density measurements were made of the left and right hemispheres over three
successive sections, which were anatomically matched across animals
according to the atlas of Franklin and Paxinos (1997). Background
optical density was subtracted from each image. Each mean ± SEM
reported here is the averaged value of six optical density readings
(after background subtraction) for each animal. Data were analyzed by
Student's t test; statistical significance was taken as
p < 0.05.
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RESULTS |
Dbh / mice have increased susceptibility to
epileptic stimuli
Flurothyl
Dbh / mice without DOPS had significantly reduced
latencies to the first myoclonic jerk (MJ) and clonic/tonic (C/T)
seizure compared with Dbh +/ controls (Fig.
1A,B, without DOPS).
The latency to the first MJ was affected to a greater degree (46% reduction) than the latency to C/T convulsion (29% reduction). The
percent of Dbh / and Dbh +/ mice
progressing to tonic extension after a C/T seizure was identical
(45%); however, 100% of the Dbh / mice died after C/T
seizure, whereas only 60% of the Dbh +/ mice died after
tonic extension (Table 1). The higher
mortality rate of Dbh / mice was not a function of the
duration of flurothyl exposure, because the average duration of
exposure was shorter for the Dbh / than for the
Dbh +/ animals.
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Figure 1.
Responsiveness of Dbh +/ and
Dbh / mice to flurothyl-induced seizures. Latencies
(seconds) to first MJ (A) and clonic/tonic
seizure (B) recorded in Dbh +/
and Dbh / mice, with and without the administration
of DOPS. Dbh / mice without DOPS had significantly
shorter latencies to MJ (A) and C/T seizures
(B) compared with Dbh +/ mice
without DOPS (mean ± SEM; single
asterisks denote p < 0.05).
Administration of DOPS (1 mg/gm) 6 hr before flurothyl significantly
increased flurothyl latencies in the Dbh / mice
compared with the Dbh / mice without DOPS for both
MJ (A) and C/T seizures (B)
(mean ± SEM; double asterisks denote
p < 0.05). Latencies to both MJ and C/T
convulsions in Dbh / mice with DOPS were not
significantly different compared with those in Dbh +/
mice with DOPS.
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NE levels are partially restored in the CNS of Dbh /
mice by the administration of DOPS (Thomas et al., 1998).
Administration of DOPS to Dbh / mice significantly
lengthened the latency to the first MJ and C/T convulsion (Fig.
1A,B with DOPS); latencies to MJ and C/T convulsions
in Dbh / mice with DOPS were not statistically different
from latencies in Dbh +/ mice with DOPS. Administration of
DOPS to Dbh +/ mice did not significantly alter the
latency to the first MJ but significantly increased the latency time to C/T seizures (Fig. 1). Administration of DOPS to Dbh /
and Dbh +/ mice did not affect the number of animals
progressing to tonic extension but did reduce the number of animals
dying after tonic extension in both groups (Table 1).
Pentylenetetrazol
Dbh / and Dbh +/ mice were challenged
with PTZ at 30 and 40 mg/kg, and the latencies (seconds) to the
first MJ, forelimb clonus (FC), and C/T were measured (Fig.
2). PTZ (40 mg/kg) induced generalized
seizures in all Dbh / mice (eight of eight) but in only four of seven Dbh +/ mice. Latencies to MJ, FC,
and C/T seizures in Dbh / mice were significantly
shorter than those in Dbh +/ mice (Fig.
2A). The percent of animals exhibiting tonic extension was greater in Dbh / mice (100%) than in
Dbh +/ mice (29%); however, for both genotypes, all
animals exhibiting tonic extension died (Fig.
2A).
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Figure 2.
Responsiveness of Dbh +/ and
Dbh / mice to PTZ injections at 40 mg/kg
(A) and 30 mg/kg (B).
Left, Graphs show seizure latencies (seconds) to the
first myoclonic jerk, forelimb clonus, and clonic/tonic seizure in
Dbh / and Dbh +/ mice. At both PTZ
concentrations Dbh / mice had significantly shorter
latencies compared with those in Dbh +/ mice
(mean ± SEM; single asterisk
denotes p < 0.01; double
asterisks denote p < 0.001).
Right, Graphs show the percentage of animals progressing
to tonic extension and the percentage of animals that died after tonic
extension (mortality).
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PTZ (30 mg/kg) induced C/T seizures in 8 of 10 Dbh /
mice and in 2 of 9 Dbh +/ mice. Of these animals
exhibiting seizures, the Dbh / mice had significantly
shorter latencies to the first MJ, FC, and C/T seizures than did
Dbh +/ mice (Fig. 2B). Again 100% of
the Dbh / mice that exhibited seizure activity
progressed to tonic extension and death; however, only 11% of the
Dbh +/ mice that exhibited seizure activity had tonic
extension, and of those, only 44% died.
Kainic acid
Kainic acid (KA; 20 mg/kg) induced some seizure behavior (i.e.,
staring, head nodding, and forelimb clonus) in most animals in both
groups; however KA induced generalized C/T convulsions in 100% of the
Dbh / mice (eight of eight) but in only 38% of the
Dbh +/ mice (three of eight). Of the animals showing C/T convulsions, Dbh / mice had a significantly shorter
latency to generalized seizure (1587 ± 188 sec) than did
Dbh +/ mice (2243 ± 120 sec). The Dbh
/ mice also exhibited enhanced sensitivity to KA compared with
Dbh +/ mice; 50% of the Dbh / mice died after the KA-induced seizure, whereas none of the Dbh +/
mice died.
Audiogenic seizures
The Dbh / mice were more sensitive to the acoustic
stimuli than were Dbh +/ mice, in that 50% (5 of 10) of
the Dbh / mice exhibited a generalized seizure during
the sound stimulus, whereas only 11% (1 of 9) of the Dbh
+/ exhibited a generalized convulsion. Seizures were initiated
shortly after onset of the sound (latencies between 3 and 12 sec) and
manifested initially as jumping behavior that progressed quickly to
explosive running-bouncing activity and finally to tonic extension and
death. Sensitivity to sound-induced seizure was identical between the
groups of animals; all animals (i.e., in both Dbh / and
Dbh +/ groups) that exhibited a sound-induced generalized
seizure died.
Dbh / mice have increased c-fos mRNA associated
with flurothyl-induced seizures
The animals that survived flurothyl-induced seizures
[Dbh +/ (n = 8) and Dbh /
(n = 6) without DOPS; Dbh +/
(n = 8) and Dbh / (n = 9) with DOPS] were killed 1 hr after C/T seizures to measure c-fos
mRNA expression. Seizure-induced c-fos mRNA expression was quantitated
in the neocortex, amygdala, and hippocampus [CA1, CA3, and dentate
gyrus (DG)] [see Figs. 3 (for
representative autoradiograms), 4 (for
quantitative comparisons)].
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Figure 3.
Representative autoradiograms of c-fos mRNA
expression after flurothyl-induced seizures. A,
C, Flurothyl-induced c-fos mRNA expression in
Dbh +/ mice without (A) and with
(C) DOPS (1 mg/gm). B, D,
Flurothyl-induced c-fos mRNA expression in Dbh /
mice without (B) and with
(D) DOPS (1 mg/gm). Note the higher c-fos mRNA
expression in Dbh / animals without DOPS
(B). DOPS (1 mg/gm) administration not only
reduces c-fos mRNA expression of Dbh / mice (compare
B with D) but also normalizes c-fos mRNA
expression in Dbh / mice relative to that in
Dbh +/ mice (compare C with
D). Scale bar, 2 mm. N,
Neocortex.
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Figure 4.
Quantification of flurothyl seizure-associated
c-fos mRNA expression in the cortex, hippocampus (CA1, CA3, and DG),
and amygdala in Dbh / and Dbh +/
mice, with and without DOPS (1 mg/gm). Flurothyl seizure-associated
c-fos mRNA expression was significantly higher in Dbh / mice without DOPS than
in Dbh +/ mice without DOPS in all regions but the
dentate gyrus (mean ± SEM; asterisks denote
p < 0.01). DOPS (1 mg/gm) administration to
Dbh / mice significantly reduced c-fos mRNA
expression in all regions but the dentate gyrus (mean ± SEM;
asterisks denote p < 0.05); c-fos
mRNA expression in Dbh / mice and Dbh
+/ mice with DOPS was not significantly different. Basal c-fos mRNA
expression in Dbh / mice is not significantly
different from basal c-fos mRNA expression in Dbh +/
mice (data not shown).
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In the neocortex, Dbh / mice had significantly greater
seizure-induced c-fos mRNA expression than did the Dbh +/
mice, even though the two different genotypes had similar
seizure-induced behavior (generalized seizures). Administration of DOPS
to Dbh / mice reduced seizure-associated c-fos mRNA
expression to a level comparable with that seen in the neocortex of
Dbh +/ mice, without or with DOPS (Fig. 4). Administration
of DOPS to Dbh +/ mice did not alter seizure-associated
c-fos mRNA expression in the neocortex.
Similar results were obtained in the hippocampal CA1 and CA3 regions
and the amygdala. Flurothyl seizure-associated c-fos mRNA expression in
Dbh / mice was significantly higher than that in
Dbh +/ mice in CA1 and CA3 regions and the amygdala. Administration of DOPS to Dbh / mice significantly
reduced the flurothyl seizure-associated c-fos mRNA expression (to the
level observed in Dbh +/ mice with DOPS). Flurothyl
seizure-associated c-fos mRNA expression in Dbh +/ mice
was not significantly changed by DOPS pretreatment. The only region
where c-fos mRNA expression was not significantly different between
Dbh / and Dbh +/ mice was in the DG.
Administration of DOPS to both genotypes also had no effect on
flurothyl-induced c-fos mRNA expression in the DG. Because the
Dbh / mice had elevated seizure-associated c-fos mRNA
expression in the neocortex, hippocampal CA1 and CA3, and amygdala,
basal c-fos mRNA was measured in Dbh +/ and Dbh
/ mice. Basal c-fos mRNA expression in Dbh / mice
was not significantly different from that in Dbh +/ mice
(data not shown).
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DISCUSSION |
These studies provide evidence that endogenous NE exerts a
profound inhibitory effect on seizure induction. The enhanced
susceptibility of Dbh / mice to such a diverse set of
seizure-inducing stimuli (convulsant stimuli potentially acting at
excitatory or inhibitory receptors, sodium channels, and brainstem
activation) (Olney et al., 1974; Schwob et al., 1980; Woodbury,
1980; Browning, 1985; Snead, 1992) suggests a "global" suppressive
action of NE. The loss of NE's inhibitory action in Dbh
/ mice is also associated with increased c-fos mRNA expression
after flurothyl-induced seizures. LC axons have a high degree of
collateralization, and a single neuron can innervate several distant
regions (Fallon and Loughlin, 1982; Loughlin et al., 1982). This
diffuse noradrenergic innervation pattern would allow NE release from
LC terminals to suppress neuronal activity throughout the brain,
including regions such as the cortex and hippocampus that are important
in regulating seizures. Our studies support the hypothesis that there
is an inverse relationship between the release of NE and seizure
susceptibility; i.e., reducing NE release increases seizure
susceptibility and increasing NE release has a protective effect
against seizures.
Although many studies have implicated NE as an endogenous
neuromodulator of seizure activity (Chen et al., 1954; Arnold et al.,
1973; Libet et al., 1977; Jerlicz et al., 1978; Mason and Corcoran,
1979; Snead, 1987; Trottier et al., 1988; Turski et al., 1989; Sullivan
and Osorio, 1991; Mishra et al., 1994), the evidence has not always
been consistent. The data presented here demonstrate that a selective
loss of NE from noradrenergic terminals is proconvulsant. One could
argue that the NE deficiency in a knock-out mouse is not definitive
because developmental changes associated with the deletion of the
Dbh gene might contribute to the seizure-susceptibility
phenotype seen in the Dbh / mice. However, we have also
shown that increasing NE content in the CNS with DOPS administration
(Thomas et al., 1998) can normalize seizure susceptibility of
Dbh / mice. The ability of DOPS to rescue Dbh
/ mice has also been demonstrated with most other behavioral and
physiological deficiencies in these mice (Thomas et al., 1995; Thomas
and Palmiter, 1997a,b,c). DOPS rescue of noradrenergic function in the
Dbh / mice suggests that there is a normal anatomical
development of the "noradrenergic" system during gestation in these
animals; this prediction of a normal pattern of noradrenergic terminals
in Dbh / mice has been confirmed in studies of NE
transporter-binding sites (D. Weinshenker, unpublished observation). If
one assumes a normal organization of noradrenergic terminals in
Dbh / mice, the conversion of DOPS to NE by
L-aromatic amino acid decarboxylase could
theoretically restore NE at appropriate terminals.
The absence of NE in Dbh / mice resulted in their
greater sensitivity (i.e., enhanced seizure severity and higher
mortality) to convulsant stimuli than that in Dbh +/
animals. After PTZ- and kainic acid-induced seizures, a higher
percentage of the Dbh / mice progressed to tonic
extension and death. A similar finding was observed with
flurothyl-induced seizures; although the percentage of animals
progressing to tonic extension was the same for Dbh +/ and
Dbh / mice without DOPS, more Dbh / mice
than Dbh +/ mice died after tonic extension.
Administration of DOPS to both Dbh +/ and Dbh
/ mice had little effect on seizure severity but reduced the number
of animals that died after tonic extension, especially in
Dbh / mice. These results reflect the ability of DOPS to
reverse the higher sensitivity of Dbh / mice to
flurothyl-induced seizures. The increased survival of DOPS-treated
Dbh +/ mice may be caused by an elevation in NE content
above a normal catecholamine content (Thomas et al., 1998).
Associated with the enhanced susceptibility to convulsant stimuli in
Dbh / mice is an elevation in seizure-induced c-fos mRNA
expression in the cortex, hippocampus (CA1 and CA3), and amygdala. The
immediate early gene c-fos has long been considered a marker of
neuronal activity (Dragunow and Robertson, 1987; Morgan et al.,
1987; Sonnenberg et al., 1989; Morgan and Curran, 1991). A correlation
of seizure severity and c-fos expression has been observed with
different convulsant stimuli (White and Price, 1993; Szot et al., 1997;
Robbins et al., 1998). The enhanced seizure-induced c-fos mRNA
expression in Dbh / mice is not a function of an elevated basal c-fos state, because basal c-fos mRNA expression in
Dbh / mice is not different from that in Dbh
+/ mice. This enhanced c-fos mRNA expression in the CNS of
Dbh / mice was measured in animals with a consistent
behavioral seizure phenotype, suggesting a relationship between c-fos
mRNA expression and seizure threshold. Acute DOPS administration
normalized the seizure-associated c-fos mRNA expression in
Dbh / mice. We conclude that the ability of the
noradrenergic system to regulate seizure activity is a direct result of
NE-mediated suppression of CNS excitability in such regions as the
neocortex, hippocampus, and amygdala.
The ability of NE to have an inhibitory effect on seizures seems
inconsistent with its general role on the arousal state of an animal.
Noradrenergic neurons are active in awake animals but quiescent during
sleep (Jouvet, 1969; Hobson et al., 1975; Aston-Jones and Bloom, 1981;
Robbins, 1984). Basal c-fos mRNA expression in the cortex corresponds
to the arousal state of the rat (Cirelli et al., 1996). When
noradrenergic neurons in the LC were destroyed with 6-hydroxydopamine,
the amount of basal c-fos mRNA expression in the cortex of the awake
animal was reduced to levels comparable with that in an animal during
sleep (Cirelli et al., 1996). Although these studies suggest a
relationship between basal c-fos mRNA expression and NE, our study
failed to find a change in basal c-fos mRNA expression in
Dbh / mice relative to Dbh +/ mice. This
difference emphasizes the gross effects of lesioning noradrenergic neurons, which results not only in the loss of NE but also affects the
level of all neurotransmitters coreleased with NE. These cotransmitters released with NE may contribute to the basal excitability of the neurons.
The dual action of NE as an inhibitory and excitatory neurotransmitter
can be attributed to the large diversity of noradrenergic receptors.
Iontophoretic application of NE to neocortex or hippocampus results in
both excitatory and inhibitory responses (Szabadi, 1979; Langmoen et
al., 1981; Nishi et al., 1981; Segal, 1981; Madison and Nicoll, 1986;
Waterhouse, 1986; Stanton, 1992). The excitatory response of NE appears
to be mediated via the -receptors and/or 1-ARs, whereas the
inhibitory response is mediated via the 2-ARs (Curet and
deMontigny, 1988; Parfitt et al., 1988; Licata et al., 1993).
This dual action of NE on neuronal activity is apparent when synaptic
NE content is elevated with NE reuptake blockers; these agents do not
alter the animal's susceptibility to convulsant stimuli (Kleinrok et
al., 1991; Yacobi and Burnham, 1991). We postulate that the
anticonvulsant action of NE is mediated via 2-ARs. Indeed, agonists
selective for the 2-ARs have been shown to exert anticonvulsant
effects against audiogenic seizures in mice, as well as against PTZ-,
kainic acid-, and bicuculline-induced seizures; 2-AR antagonists
have the reverse effect (Papanicolaou et al., 1982; Baran et al., 1985;
Loscher and Czuczwar, 1987; Fletcher and Forster, 1988; Jackson et al.,
1991). However, it has not been determined whether the anticonvulsant
effect of 2-AR agonists is mediated via the pre- or postsynaptic
receptors. A recently developed transgenic mouse with nonfunctional
2A-ARs (MacMillan et al., 1996) responded to a kindling paradigm (a
process of repetitively applied stimuli resulting in generalized
seizures) similarly to wild-type mice treated with an 2-AR
antagonist (Janumpalli et al., 1998). Although the Dbh
/ mice are not the same as the 2A-AR mutant, the combined
results provide compelling evidence that NE acting at least partially
via inhibitory postsynaptic 2-ARs dampens seizure excitability. The
lack of spontaneous seizure activity in Dbh / mice
suggests that NE release may only become important under conditions of
high activity (e.g., seizures) when the LC is sufficiently activated;
i.e., NE serves as a potent modulator of excitability.
In conclusion, the data presented here show unambiguously that NE is
capable of modulating seizure activity induced by different convulsant
stimuli. The pervasive inhibitory action of NE on excitability is
reflected in the increased seizure-associated c-fos mRNA expression in
the Dbh / mice. Because galanin and NPY are also
inhibitory neuromodulators that are coreleased from the same terminals
as NE, it seems that the noradrenergic projection system may use multiple neurotransmitters to dampen excitability. Because of this
complexity, the Dbh / mice provide an especially useful and new system to examine the pathways through which NE regulates seizure activity.
| |
FOOTNOTES |
Received July 7, 1999; revised Sept. 28, 1999; accepted Sept. 30, 1999.
This work was supported by the National Alliance for Research on
Schizophrenia and Depression (P.S.), the Department of Veterans Affairs
(P.S.), National Institutes of Health Grant NS-18895 (P.A.S.), and the
Howard Hughes Medical Institute (D.W. and N.C.R). We thank Sumitomo
Pharmaceuticals for their generous donation of DOPS.
P.S. and D.W. contributed equally to this work.
Correspondence should be addressed to Dr. Patricia Szot, Geriatric
Research, Education, and Clinical Center (182B), Veterans Affairs
Medical Center, 1660 South Columbian Way, Seattle, WA 98108. E-mail:
szot{at}u.washington.edu.
| |
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ABSTRACT
INTRODUCTION
