0022-3565/01/2983-1042–1048$3.
00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 298:1042–1048, 2001
␣1 and ␤2 Adrenoreceptor Agonists Inhibit Pentylenetetrazole-
Induced Seizures in Mice Lacking Norepinephrine
DAVID WEINSHENKER, PATRICIA SZOT, NICOLE S. MILLER, and RICHARD D. PALMITER
Howard Hughes Medical Institute (D.W., N.S.M., R.D.P.) and Departments of Biochemistry (D.W.) and Psychiatry and Behavioral Sciences
(P.S.), University of Washington, Seattle, Washington; and Geriatric Research, Education, Clinical Center (GRECC), Puget Sound Health Care
System, Seattle, Washington (P.S.)
Received April 5, 2001; accepted May 25, 2001 This paper is available online at http://jpet.aspetjournals.org
ABSTRACT
It has been known for many years that norepinephrine (NE) is a
potent endogenous anticonvulsant, yet there is confusion as to
which receptor(s) mediate this effect. This is probably due to
multiple factors, including the importance of distinct signaling
pathways for different seizure paradigms, a lack of comprehen-
sive pharmacological studies, and difficulty in interpreting ex-
isting pharmacological results due to the presence of endoge-
nous NE. We sought to circumvent these problems by testing
the anticonvulsant activity of selective agonists for most known
adrenoreceptors (ARs) in dopamine ␤-hydroxylase knockout
(Dbh ⫺/⫺) mice that lack endogenous NE. Dbh ⫺/⫺ mice are
hypersensitive to pentylenetetrazole (PTZ)-induced seizures,
demonstrating that endogenous NE inhibits PTZ-induced sei-
The importance of endogenous norepinephrine (NE) as an
anticonvulsant neurotransmitter is well established. For ex-
ample, specific lesions of the central noradrenergic system
using 6-hydroxydopamine (Corcoran et al., 1974) or N-(2-chlo-
roethyl)-N-2-bromobenzylamine (Carre and Harley, 1986) re-
sult in increased seizure sensitivity. Conversely, stimulation of
the locus coeruleus, the primary central noradrenergic nucleus,
significantly inhibits seizures (Weiss et al., 1990). More
recently, antiepileptic therapies used clinically have been
shown to either increase central NE content or require an
intact noradrenergic system for their efficacy (Waller and
Buterbaugh, 1985; Baf et al., 1994; Krahl et al., 1998; P.
Szot, D. Weinshenker, J. M. Rho, T. W. Storey, and P. A.
Schwartzkroin, unpublished data). Despite the long-stand-
ing interest and experimentation along these lines, it is
still unclear exactly how NE inhibits seizure activity.
There are three primary reasons for this confusion.
First, the noradrenergic signaling system is extremely
complex; three distinct classes of receptor with multiple sub-
types have been cloned, and each class activates different G
D.W. and N.S.M. were supported by the Howard Hughes Medical Institute.
P.S. was supported by the National Alliance for Research on Schizophrenia
and Depression and the Department of Veterans Affairs.
ABBREVIATIONS: NE, norepinephrine; AR, adrenoreceptor; PTZ, pentylenetetrazole; Dbh, dopamine ␤-hydroxylase; MJ, myoclonic jerk; C/T,
clonic/tonic; DMSO, dimethyl sulfoxide.
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zures in the wild type. Pretreatment of Dbh ⫺/⫺ mice with an
␣1AR or ␤2AR, but not an ␣2AR or ␤1AR agonist significantly
protected against PTZ-induced seizures. In contrast, only the
␤2AR agonist showed anticonvulsant activity in heterozygous
controls. Furthermore, an ␣1AR antagonist exacerbated PTZ-
induced seizures in control mice, whereas a ␤2AR antagonist
had no effect. We conclude that activation of the ␣1AR is
primarily responsible for the anticonvulsant activity of endoge-
nous NE in the murine PTZ model of epilepsy. Endogenous NE
probably does not activate the ␤2AR under these conditions,
but exogenous activation of the ␤2AR produces an anticonvul-
sant effect.
proteins. The ␣1 class of adrenoreceptors (ARs) is linked to
Go/Gq and activates phospholipase C and intracellular Ca2⫹
release. ␣2ARs are linked to Gi and inhibit adenylate cyclase.
␤ARs (␤1AR and ␤2AR) are linked to Gs and activate adenyl-
ate cyclase (for review, see Cooper et al., 1996). All of these
receptor classes are widely distributed throughout the brain
and are found in regions implicated in regulating seizures
such as the hippocampus, cortex, and amygdala. Differences
in receptor distribution between animal species, strain, and
the brain region(s) affected by seizure-inducing paradigms
probably contribute to the variety of ARs shown to possess
anticonvulsant activity (Papanicolaou et al., 1982; Lints and
Nyquist-Battie, 1985; Neuman, 1986; Löscher and Czuczwar,
1987; Ferraro et al., 1994; Yan et al., 1998). Second, despite
this complexity, many seizure susceptibility studies have not
been pharmacologically comprehensive for any given seizure
paradigm. For example, data have been presented exclu-
sively on nonselective ␤AR antagonists (Lints and Nyquist-
Battie, 1985), nonselective ␤AR agonists and antagonists
(Ferraro et al., 1994), and ␣AR agonists and antagonists
(Löscher and Czuczwar, 1987; Tsuda et al., 1990). In only a
few studies have compounds that target both ␣ARs and ␤ARs
been tested (Neuman, 1986; Gellman et al., 1987; Micheletti

et al., 1987), and even these were somewhat limited in their
scope.
Third, although the ␣2AR receptor is probably the most
promiscuous anticonvulsant receptor in terms of efficacy
across species, strain, and seizure paradigm (Papanicolaou et
al., 1982; Baran et al., 1985; Scotti de Carolis et al., 1986;
Löscher and Czuczwar, 1987; Jackson et al., 1991), procon-
vulsant effects have also been reported (Oishi et al., 1979;
Löscher and Czuczwar, 1987; Wu et al., 1987). In addition,
there are inherent difficulties interpreting any of these re-
sults. Three different subtypes of ␣2AR exist (␣2A, ␣2B, and
␣2C) and they are localized both pre- and postsynaptically.
Activation of presynaptic ␣2AR autoreceptors decreases nor-
adrenergic firing and NE release (L’Heureux et al., 1986;
Jorm and Stamford, 1993), while activation of postsynaptic
␣2ARs mimics the effect of released NE on target cells ex-
pressing these receptors and inhibits their firing (Gobert et
al., 1998). Because available ␣2AR agonists cannot distin-
guish between ␣2AR subtypes or pre- versus postsynaptic
␣2ARs, it is nearly impossible to interpret the effect of ␣2AR
agonists. In addition, there is some evidence that activation
of presynaptic ␤ARs facilitate central NE release (Murugaiah
and O’Donnell (1995).
To address these issues, we have tested the effects of sub-
type-selective agonists and antagonists for most known ARs
on susceptibility to pentylenetetrazole (PTZ)-induced sei-
zures in dopamine ␤-hydroxylase knockout (Dbh ⫺/⫺) mice
that are deficient in NE synthesis and lack NE, thereby
isolating the postsynaptic effects of these compounds and
eliminating any influence on NE release. Dbh ⫺/⫺ mice
have increased susceptibility to PTZ-induced seizures, dem-
onstrating that endogenous NE is anticonvulsant in this
model of epilepsy (Szot et al., 1999). Both anticonvulsant
(Löscher and Czuczwar, 1987; Amabeoku et al., 1994) and
proconvulsant (Oishi et al., 1979; Fletcher and Forster, 1988)
effects of clonidine on PTZ-induced seizures in mice have
been reported. We predicted that the effects of clonidine
would be abolished in Dbh ⫺/⫺ mice if it acts via presynaptic
␣2ARs, but Dbh ⫺/⫺ mice would still be affected if the
postsynaptic ␣2ARs are involved.
Materials and Methods
Animals. Dbh knockout (Dbh ⫺/⫺) mice, maintained on a 129/
SvEv and C57BL/6J hybrid background, were developed and gener-
ated as described (Thomas et al., 1998). Mice were reared in a
specific pathogen-free facility with a 12-h light/dark cycle at the
University of Washington (Seattle, WA), but moved to a conventional
facility for seizure experiments. Mice between 3 and 6 months of age
were used for all experiments, and food and water were available ad
libitum.
Dbh ⫺/⫺ mice were identified by the delayed growth and ptosis
phenotype, which is 100% correlated with the Dbh ⫺/⫺ genotype
(data not shown). A subset of genotypes was confirmed by polymer-
ase chain reaction. Dbh ⫹/⫺ mice have normal levels of epinephrine
and NE and are indistinguishable from wild-type littermates for all
previously tested behaviors, including seizure susceptibility (Thom-
as et al., 1998; Szot et al., 1999). Therefore, heterozygous (Dbh ⫹/⫺)
littermates were used as controls for all experiments in this study.
Experimental protocols were approved by the animal care committee
at the University of Washington and meet the guidelines of the
American Association for Accreditation of Laboratory Animal Care.
Seizure Induction. Seizures were induced with an i.p. injection
of PTZ dissolved in water. Because Dbh ⫺/⫺ mice are more sensitive
␣1AR and ␤2AR Agonists Inhibit PTZ Seizures 1043
Fig. 1. Susceptibility of Dbh ⫹/⫺ and Dbh ⫺/⫺ mice to PTZ-induced
seizures. Shown are latencies to first MJ and C/T seizure. These data are
a compilation of results with water and vehicle-injected mice followed by
administration of 40 mg/kg PTZ from Figs. 2 through 4 (Dbh ⫹/⫺, n ⫽
49; Dbh ⫺/⫺, n ⫽ 67). ****P ⬍ 0.0001 compared with Dbh ⫹/⫺.
to PTZ-induced seizures (Fig. 1; Szot et al., 1999), doses of PTZ were
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adjusted based on genotype in many experiments to elicit seizures of
similar severity in Dbh ⫹/⫺ and Dbh ⫺/⫺ mice. PTZ was admin-
istered at a dose of 25, 30, or 40 mg/kg (6.25, 7.5, or 10 mg/ml) for Dbh
⫺/⫺ mice and 40 or 50 mg/kg (10 or 12.5 mg/ml) for Dbh ⫹/⫺ mice
in a volume of 4 ml/kg. The lower doses of PTZ for each genotype,
which produced mild seizures, were used when testing a drug that
had a proconvulsant effect based on pilot experiments, while the
higher doses produced severe seizures and were used when testing
anticonvulsant compounds. Mice were placed in a clear Plexiglas
chamber and closely observed for 10 min. This observation time was
chosen because mice that displayed seizure activity did so within the
first few minutes after PTZ administration and were typically pos-
tictal by 10 min. Latency to first myoclonic jerk (MJ) and clonic/tonic
(C/T) seizure was recorded. Animals that did not experience one of
these behavioral seizure landmarks were assigned a latency of 600 s
for that measurement.
To test the effects of AR agonists and antagonists, clonidine,
yohimbine, phenylephrine, cirazoline, isoproterenol, dobutamine, al-
buterol, prazosin, propanolol, ICI-118,551, or idazoxan (Sigma, St.
Louis, MO) were administered intraperitoneally in a volume of 4
ml/kg 30 min prior to seizure induction with PTZ. To test the ability
of an antagonist to block the anticonvulsant activity of an agonist,
the antagonist was administered 30 min prior to the agonist. All
drugs were dissolved in water with the exception of prazosin, which
was dissolved in a mixture of 1.5% DMSO and 1.5% Cremaphor EL
(Sigma). For prazosin experiments, the DMSO/Cremaphor EL solu-
tion was used as a vehicle control. Water was used as a vehicle
control for all other experiments. Drug doses were based on those
used in the murine seizure literature when available, and on pub-
lished rat data or pilot experiments when a compound had not been
previously test in mice.
Data were analyzed using Student’s t tests for comparing two
groups with means of equivalent variance, Mann-Whitney U tests for
comparing two groups with means of nonequivalent variance, and
analysis of variance followed by Newman-Keuls post hoc tests for
comparing means from more than two groups. P ⬍ 0.05 was consid-
ered significant.
Results
Dbh ⴚ/ⴚ Mice Are Hypersensitive to PTZ-Induced
Seizures. We previously reported that Dbh ⫺/⫺ mice have
shorter latencies to first MJ and C/T seizure than Dbh ⫹/⫺
mice following PTZ administration (Szot et al., 1999). We
repeated this experiment with a larger number of mice and
obtained the same result (Fig. 1), demonstrating that a lack
of endogenous NE results in increased sensitivity to PTZ-
induced seizures.
1044 Weinshenker et al.
␣2AR Agonists Act via Autoreceptors to Exacerbate
PTZ-Induced Seizures. To determine whether ␣2AR ago-
nists increased or decreased sensitivity to PTZ-induced sei-
zures, we treated Dbh ⫹/⫺ mice with the ␣2AR agonist
clonidine prior to PTZ administration. Clonidine (0.1 mg/kg)
had a proconvulsant effect that was most pronounced for
latency to MJ (Fig. 2A). To determine whether this procon-
vulsant effect was mediated by an inhibition of NE release
via presynaptic autoreceptors or via actions on postsynaptic
heteroreceptors, we tested the effect of clonidine on PTZ-
induced seizures in Dbh ⫺/⫺ mice. Clonidine did not alter
seizure latency in Dbh ⫺/⫺ mice in response to 30 mg/kg
PTZ (Fig. 2A) or 25 mg/kg PTZ (latency to MJ: vehicle ⫽
317 ⫾ 73 s, 0.1 mg/kg clonidine ⫽ 265 ⫾ 29 s, P ⫽ 0.85; no C/T
seizures observed), suggesting that the proconvulsant effect
seen in control mice resulted from decreased NE release
mediated by activation of presynaptic ␣2AR autoreceptors.
Clonidine treatment caused piloerection in both genotypes
and rescued the bilateral ptosis phenotype of Dbh ⫺/⫺ mice,
demonstrating that the drug possessed biological activity at
this dose in the knockouts (data not shown). Blockade of
␣2ARs with yohimbine (10 mg/kg) slightly increased seizure
latency in both genotypes, but the effect was not significant
(Fig. 2B). The weak anticonvulsant effect of yohimbine may
have been nonspecific and caused by the significant sedation
observed with this treatment. The ␣2AR antagonist idazoxan
(3 mg/kg), which lacks the sedative effects of yohimbine, also
did not have a significant effect (data not shown). These
results suggest that while exogenous activation of ␣2ARs can
exacerbate PTZ-induced seizures, activation of ␣2ARs by en-
dogenous NE is not involved under these conditions.
Activation of Central ␣1ARs Inhibits PTZ-Induced
Seizures in Dbh ⴚ/ⴚ Mice. While clonidine is a fairly
Fig. 2. Effects of ␣2AR-acting drugs on PTZ-induced seizure susceptibil-
ity. Shown are latencies to first MJ and C/T seizure. A, water (Dbh ⫹/⫺,
n ⫽ 13; Dbh ⫺/⫺, n ⫽ 14) or clonidine (0.1 mg/kg: Dbh ⫹/⫺, n ⫽ 14; Dbh
⫺/⫺, n ⫽ 14) was administered 30 min prior to PTZ (40 mg/kg for Dbh
⫹/⫺, 30 mg/kg for Dbh ⫺/⫺). *P ⬍ 0.05 compared with water control. B,
water (Dbh ⫹/⫺, n ⫽ 12; Dbh ⫺/⫺, n ⫽ 8) or yohimbine (10 mg/kg: Dbh
⫹/⫺, n ⫽ 12; Dbh ⫺/⫺, n ⫽ 8) was administered 30 min prior to PTZ (50
mg/kg for Dbh ⫹/⫺, 40 mg/kg for Dbh ⫺/⫺).
selective ␣2AR agonist, it has some activity at ␣1ARs at high
doses (Wu et al., 1987). We considered the possibility that our
results with clonidine were due to concurrent activation of
␣1ARs, but rejected this hypothesis based on the results of
two experiments. First, decreasing the dose of clonidine to 1
␮g/kg failed to reveal an anticonvulsant effect (data not
shown). This dose was chosen because it is maximally effec-
tive at inhibiting PTZ-induced seizures in normal rats and
electrically induced seizures in rats with 6-hydroxydopamine
lesions of their noradrenergic systems (Dalton et al., 1985).
Second, cirazoline (0.2 mg/kg), a selective ␣1AR agonist, sig-
nificantly increased latencies to MJ and C/T seizures in Dbh
⫺/⫺ mice (Fig. 3A). While eight of nine vehicle-treated Dbh
⫺/⫺ mice progressed to a C/T seizure at this dose of PTZ,
only 2 of 10 cirazoline-treated Dbh ⫺/⫺ mice had seizures of
this severity. Similar effects were obtained with 0.1 and 0.5
mg/kg cirazoline (data not shown). This anticonvulsant effect
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was blocked by pretreatment with prazosin (1 mg/kg), a se-
lective ␣1AR antagonist, further verifying the importance of
␣1ARs (Fig. 3B). While cirazoline did not have a significant
anticonvulsant effect on Dbh ⫹/⫺ controls (Fig. 3A), prazo-
sin alone (1 mg/kg) had a proconvulsant effect (Fig. 3C),
reducing latencies to MJ and C/T seizures. These data sug-
gest that the inhibition of seizures by endogenous NE in
normal animals involves ␣1ARs.
Because ␣1AR agonists have profound effects on the pe-
ripheral nervous system, we determined whether the anti-
convulsant effect of cirazoline was mediated by central or
peripheral ␣1 receptors. In contrast to cirazoline, phenyleph-
rine (3 mg/kg), an ␣1AR agonist that cannot cross the blood-
brain barrier, failed to inhibit seizures in Dbh ⫺/⫺ mice (Fig.
3D) despite having qualitatively similar effects on ptosis and
piloerection (data not shown). These results suggest that Dbh
⫺/⫺ mice are more susceptible to PTZ-induced seizures at
least in part because of a lack of central ␣1AR signaling.
␤2AR Signaling Inhibits PTZ-Induced Seizures. To
investigate the contribution of ␤AR signaling to the anticon-
vulsant effect of NE, we treated mice with isoproterenol, a
nonselective ␤AR agonist. We found that isoproterenol (10
mg/kg) increased latencies to PTZ-induced seizures in both
Dbh ⫺/⫺ and Dbh ⫹/⫺ mice (Fig. 4A). To determine which
␤AR subtype was responsible for the anticonvulsant effect of
isoproterenol, mice were treated with a preferential ␤1AR
(dobutamine) or a preferential ␤2AR (albuterol) agonist prior
to PTZ administration. While dobutamine (10 mg/kg) did not
significantly affect seizure susceptibility in Dbh ⫺/⫺ mice,
albuterol (10 mg/kg) prolonged the latency to both MJ and
C/T seizures (Fig. 4B). Nearly all water-treated Dbh ⫺/⫺
mice (22 of 23) progressed to C/T seizures, while only 6 of 22
albuterol-treated Dbh ⫺/⫺ mice had seizures of that severity
(data from Fig. 4, B and C, combined). Albuterol also signif-
icantly protected Dbh ⫹/⫺ control mice (Fig. 4B). Propanolol
(10 mg/kg), a nonselective ␤AR antagonist, blocked the anti-
convulsant effect of albuterol in terms of latency to MJ but
not C/T seizure in Dbh ⫺/⫺ mice (Fig. 4C). To determine
whether the inability of propanolol to completely block the
anticonvulsant effect of albuterol was due to blockade of
␤1ARs as well as ␤2ARs, we tested the ␤2AR-selective antag-
onist ICI-118,551. ICI-118,551 (10 mg/kg) abolished the an-
ticonvulsant effect of albuterol on MJ, but only partially
blocked its effect on C/T seizures (Fig. 4C). Nadolol (10 mg/
kg), a nonselective ␤AR antagonist that cannot cross the
 ␣1AR and ␤2AR Agonists Inhibit PTZ Seizures 1045

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Fig. 3. Effects of ␣1AR-acting drugs on PTZ-induced seizure susceptibility. Shown are latencies to first MJ and C/T seizure. A, water (n ⫽ 8 for each
genotype) or cirazoline (0.2 mg/kg: Dbh ⫹/⫺, n ⫽ 8; Dbh ⫺/⫺, n ⫽ 9) was administered 30 min prior to PTZ (50 mg/kg for Dbh ⫹/⫺, 40 mg/kg for
Dbh ⫺/⫺). *P ⬍ 0.05 compared with water control; **P ⬍ 0.01 compared with water control. B, vehicle (1.5% DMSO, 1.5% Cremaphor EL in water)
or prazosin (1 mg/kg) was administered 30 min prior to water or cirazoline (0.2 mg/kg; n ⫽ 10 for each drug treatment group), which was administered
to Dbh ⫺/⫺ mice 30 min prior to PTZ (40 mg/kg). *P ⬍ 0.05 compared with water control. ***P ⬍ 0.001 compared with water control. †P ⬍ 0.01
compared with cirazoline alone. C, vehicle (1.5% DMSO, 1.5% Cremaphor EL in water) or prazosin (1 mg/kg; n ⫽ 12 for each drug treatment group)
was administered to Dbh ⫹/⫺ mice 30 min prior to PTZ (40 mg/kg). *P ⬍ 0.05 compared with vehicle control; **P ⬍ 0.01 compared with vehicle control.
D, water (n ⫽ 6) or phenylephrine (3 mg/kg; n ⫽ 7) was administered to Dbh ⫺/⫺ mice 30 min prior to PTZ (40 mg/kg).

blood-brain barrier, had effects similar to ICI-118,551 (Fig.
4C). Last, neither propanolol (10, 20, or 40 mg/kg) nor ICI-
118,551 affected susceptibility to PTZ-induced seizures in
Dbh ⫹/⫺ control mice (Fig. 4D; data not shown). These
results suggest that ␤2ARs do not mediate the anticonvul-
sant effect of endogenous NE, but that exogenous activation
of ␤2ARs can inhibit PTZ-induced seizures in mice.
Discussion
The results of this study are summarized in Table 1 and
reveal three important points. First, stimulation of ␣2ARs
has a proconvulsant effect on PTZ-induced seizures. This
effect is probably mediated by the inhibition of NE release via
presynaptic ␣2 autoreceptors, because the proconvulsant ef-
fect is abolished in Dbh ⫺/⫺ mice. Second, stimulation of
either ␣1ARs or ␤2 ARs restores normal PTZ-induced seizure
susceptibility upon Dbh ⫺/⫺ mice. Third, ␣1AR but not
␤2AR antagonists confer increased seizure susceptibility
upon control mice, suggesting that endogenous NE inhibits
seizures by activating ␣1ARs in animals with normal NE
content.
Role of ␣2ARs in PTZ Seizures. The literature indicates
that ␣2ARs are the most likely receptor type to mediate the
anticonvulsant effect of endogenous NE to PTZ-induced sei-
zures in rats. Multiple independent investigators have found
that ␣2AR agonists inhibit PTZ-induced seizures (Papanico-
laou et al., 1982; Scotti de Carolis et al., 1986; Löscher and
Czuczwar, 1987), and that ␣2AR antagonists block the anti-
convulsant effect of ␣2AR agonists and potentiate PTZ-in-
duced seizures on their own (Lazarova and Samanin, 1983;
Scotti de Carolis et al., 1986). Because endogenous NE is
anticonvulsant, the interpretation of these results was that
the anticonvulsant effect of ␣2AR agonists was mediated by
postsynaptic receptors. However, one of the earliest publica-
tions on this theme reported a proconvulsant effect of
clonidine on PTZ-induced seizures in mice, and proposed that
activation of presynaptic ␣2ARs decreased NE release and
exacerbated seizures (Oishi et al., 1979). Since then, both
proconvulsant (Fletcher and Forster, 1988) and anticonvul-
sant (Löscher and Czuczwar, 1987; Amabeoku et al., 1994)
effects of clonidine in mice have been reported. We undertook
this study using Dbh ⫺/⫺ mice that lack endogenous NE to
determine whether clonidine is pro- or anticonvulsant and
whether these effects are mediated by pre- or postsynaptic
␣2ARs. We found that clonidine has a proconvulsant effect on
control mice but no effect on Dbh ⫺/⫺ mice, suggesting that
in our paradigm clonidine decreases NE release via presyn-
aptic ␣2ARs in normal animals.
Clonidine has been shown to have an anticonvulsant effect
in models where seizures were induced by kainic acid (Baran
et al., 1985), amygdala kindling (Gellman et al., 1987; Lös-
cher and Czuczwar, 1987), and air blast stimulation (Löscher
and Czuczwar, 1987). However, clonidine has exhibited pro-
convulsant effects in others, such as electroconvulsions (Lös-
cher and Czuczwar, 1987), quinolinic acid (Wu et al., 1987),
and spontaneous petit mal seizures (Micheletti et al., 1987).
1046 Weinshenker et al.

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Fig. 4. Effects of ␤AR-acting drugs on PTZ-induced seizure susceptibility. Shown are latencies to first MJ and C/T seizure. A, water (Dbh ⫹/⫺, n ⫽
10; Dbh ⫺/⫺, n ⫽ 9) or isoproterenol (10 mg/kg: Dbh ⫹/⫺, n ⫽ 10; Dbh ⫺/⫺, n ⫽ 9) was administered 30 min prior to PTZ (50 mg/kg for Dbh ⫹/⫺,
40 mg/kg for Dbh ⫺/⫺). *P ⬍ 0.05 compared with water control. **P ⬍ 0.01 compared with water control. ****P ⬍ 0.0001 compared with water control.
B, water (Dbh ⫹/⫺, n ⫽ 9; Dbh ⫺/⫺, n ⫽ 13), albuterol (10 mg/kg: Dbh ⫹/⫺, n ⫽ 9; Dbh ⫺/⫺, n ⫽ 5), or dobutamine (10 mg/kg: Dbh ⫺/⫺, n ⫽ 8)
was administered 30 min prior to PTZ (50 mg/kg for Dbh ⫹/⫺, 40 mg/kg for Dbh ⫺/⫺). **P ⬍ 0.01 compared with water control. ***P ⬍ 0.001
compared with water control. C, water, propanolol (10 mg/kg), ICI-118,551 (10 mg/kg), or nadolol (10 mg/kg) was administered 30 min prior to water
or albuterol (10 mg/kg), which was administered to Dbh ⫺/⫺ mice (water ⫹ water, n ⫽ 18; water ⫹ albuterol, n ⫽ 17; propanolol ⫹ albuterol, n ⫽
9; ICI-118,551 ⫹ albuterol, n ⫽ 12; nadolol ⫹ albuterol, n ⫽ 16) 30 min prior to PTZ (40 mg/kg). *P ⬍ 0.05 compared with water control. **P ⬍ 0.01
compared with water control. ***P ⬍ 0.001 compared with water control. †P ⬍ 0.05 compared with albuterol alone. ††P ⬍ 0.01 compared with albuterol
alone. D, water, propanolol (10 mg/kg), or ICI-118,551 (10 mg/kg) was administered to Dbh ⫹/⫺ mice (n ⫽ 10 for each drug treatment group) 30 min
prior to PTZ (40 mg/kg).

TABLE 1 esize that the proconvulsant effects seen with clonidine are
Summary of AR agonist and antagonist effects on PTZ-induced seizure mediated by ␣2AR autoreceptors, while the anticonvulsant
susceptibility
effects observed are mediated by ␣2AR heteroreceptors on
Dbh ⫺/⫺ mice are more susceptible to PTZ-induced seizures than Dbh ⫹/⫺ con-
trols, and some AR agonists and antagonists affect susceptibility differently in the target neurons.
two genotypes. 1 represents an increase in latency, 2 represents a decrease in Role of ␣1ARs in PTZ Seizures. There are three results
latency, and ⫺ represents no change in latency to PTZ-induced seizure compared
with the no drug control for each genotype. that support our conclusion that ␣1AR activation mediates
the anticonvulsant effects of endogenous NE in PTZ-induced
Dbh ⫺/⫺ Dbh ⫹/⫺
seizures. First, Dbh ⫺/⫺ mice that lack endogenous NE are
Agonists hypersensitive to PTZ-induced seizures, and administration
␣1 (Cirazoline) 1 ⫺
␣2 (Clonidine) ⫺ 2 of an ␣1AR-selective agonist can restore normal sensitivity.
␤ (Isoproterenol) 1 1 Second, inhibition of PTZ-induced seizures by cirazoline can
␤1 (Dobutamine) ⫺ ⫺ be blocked by prazosin, a selective ␣1AR antagonist. Third,
␤2 (Albuterol) 1 1
Antagonists prazosin administration in control mice results in increased
␣1 (Prazosin) NT 2 PTZ sensitivity. The inability of cirazoline to significantly
␣2 (Yohimbine) ⫺ ⫺ protect Dbh ⫹/⫺ mice may mean that ␣1ARs are already
␤ (Propanolol) NT ⫺
␤1 (Not tested) NT NT maximally activated in normal animals during seizures. Al-
␤2 (ICI-118,551) NT ⫺ though ␣1ARs have not previously been implicated in PTZ-
NT, not tested. induced seizures (Löscher and Czuczwar, 1987), ␣1 agonists
have anticonvulsant activity in many other seizure models,
In fact, clonidine has a proconvulsant effect in humans with including amygdala kindling (Löscher and Czuczwar, 1987),
intractable focal epilepsies (Kirchberger et al., 1998). One air blast stimulation (Löscher and Czuczwar, 1987), focal
explanation for these conflicting results is that these diverse cortical penicillin (Neuman, 1986), audiogenic seizures (Yan
seizure-inducing paradigms involve distinct mechanisms and et al., 1998), quinolinic acid (Wu et al., 1987), and spontane-
different brain regions, and therefore the effect of ␣2AR ago- ous petit mal seizures (Micheletti et al., 1987). Further evi-
nists and antagonists varies with ␣2AR distribution and dence that ␣1ARs mediate the anticonvulsant effect of endog-
function, species, and strain. Because endogenous NE is an- enous NE in some situations comes from studies showing
ticonvulsant in most seizure-inducing paradigms, we hypoth- changes in ␣1AR density in seizure-sensitive rats (Nicoletti et

al., 1986; Gundlach et al., 1995), mice (Jazrawi and Horton,
1986), and humans with epilepsy (Brière et al., 1986).
Role of ␤2ARs in PTZ Seizures. Our results indicate
that activation of ␤2ARs, but not ␤1ARs, inhibits PTZ-in-
duced seizures in both norepinephrine-deficient Dbh ⫺/⫺
mice and Dbh ⫹/⫺ mice, which have normal NE content.
Because mice lacking NE were affected, we conclude that
albuterol exerts its anticonvulsant action via postsynaptic
␤2ARs and does not involve an increase in NE release via
presynaptic ␤2ARs. The lack of a proconvulsant effect of the
␤AR antagonists propanolol or ICI-118,551 on Dbh ⫹/⫺ mice
suggests that ␤2ARs do not mediate the anticonvulsant effect
of endogenous NE, even though ␤2AR agonists can have an
anticonvulsant affect on mice with normal NE content. In
fact, it is unclear whether endogenously released NE typi-
cally activates ␤2ARs in the brain. For example, ␤2ARs have
a much lower affinity than ␤1ARs for NE (for review, see
Molinoff, 1984). ␤1AR but not ␤2AR antagonists affect behav-
ior (Pandey et al., 1995) and only ␤1ARs undergo changes in
response to treatments that chronically alter synaptic NE
(Minneman et al., 1982). However, exogenously supplied
␤2AR agonists affect behavior, ␤2AR density (O’Donnell,
1990), and NE release (Murugaiah and O’Donnell, 1995),
suggesting that ␤2ARs are functional when artificially stim-
ulated, as they appear to be in our study.
The anticonvulsant site of action of albuterol remains un-
resolved. While all of the ␤2AR antagonists exhibited the
ability to reverse the effect of albuterol on latency to MJ,
none were especially efficacious at blocking its effects on
generalized C/T seizures. The fact that isoproterenol and
nadolol, which are reported not to cross the blood-brain bar-
rier, had any effect suggests that albuterol is acting at least
partially via a peripheral mechanism. Because NE has pro-
found effects on the cardiovascular system, it is possible that
these drugs are exerting their effects on seizures by modu-
lating heart rate or blood pressure. However, an alternative
explanation is that these drugs may get into the brain when
administered peripherally at high doses. For example, i.p.
administration of 1 mg/kg isoproterenol results in a substan-
tial increase in phosphorylation of Ca2⫹ channels in the
central nervous system (J. Hell, personal communication).
Comparison of the anticonvulsant effects of central versus
peripheral albuterol administration will help resolve this
issue. The inability of any ␤AR antagonist to completely
block the anticonvulsant effect of albuterol may be related to
relative doses, receptor affinities, and blood-brain barrier
permeability.
Conclusions
This study comprehensively examines the anticonvulsant
pharmacology of NE for a single convulsant agent, PTZ. The
use of Dbh ⫺/⫺ mice, which lack NE, circumvents many of
the caveats in the interpretation of results obtained in the
presence of endogenous NE, and provides an example of how
a combination of genetics and pharmacology can be a power-
ful approach in the study of neurotransmitter function.
It is important to note that each class of convulsant agent
probably elicits seizures by distinct mechanisms that most
likely involve different regions of the brain. Therefore, it is
not surprising that there is not one “universal” noradrenergic
receptor that mediates the anticonvulsant effect of NE for all
␣1AR and ␤2AR Agonists Inhibit PTZ Seizures 1047
seizure models. Because Dbh ⫺/⫺ mice are also more sus-
ceptible to seizures induced by kainic acid, flurothyl, and
sound (Szot et al., 1999), it will be possible to apply the same
approach to the identification of the anticonvulsant NE re-
ceptor(s) involved in other seizure models.
Acknowledgments
We thank Sumitomo Pharmaceuticals for the generous donation of
L-threo-3,4-dihydroxyphenylserine (DOPS) and D. Kim, R. Gillis,
and M. Szczypka for critical reading of this manuscript.
References
Amabeoku G, Chikuni O and Bwakura E (1994) Gamma aminobutyric acid media-
tion of the anticonvulsant effect of clonidine on pentylenetetrazol-induced seizures
in mice. Pharmacol Res 29:273–230.
Baf MH, Subhash MN, Lakshmana KM and Rao BS (1994) Sodium valproate
induced alterations in monoamine levels in different regions of the rat brain.
Neurochem Int 24:67–72.
Baran H, Sperk G, Hortnagl H, Sapetschnig G and Hornykiewicz O (1985) Alpha
2-adrenoceptors modulate kainic acid-induced limbic seizures. Eur J Pharmacol
Downloaded from jpet.aspetjournals.org at Univ of Washington on February 14, 2012
113:263–269.
Brière R, Sherwin AL, Robitaille Y, Olivier A, Quesney LF and Reader TA (1986)
Alpha-1 adrenoceptors are decreased in human epileptic foci. Ann Neurol 19:26 –
30.
Carre G and Harley C (1986) DSP-4 treatment accelerates kindling. Exp Neurol
94:812– 816.
Cooper JR, Bloom FE and Roth RH (1996) Norepinephrine and epinephrine, in The
Biochemical Basis of Neuropharmacology (House J ed) pp 226 –292, Oxford Uni-
versity Press, New York.
Corcoran ME, Fibiger HC, McCaughran JA and Wada JA (1974) Potentiation of
amygdala kindling and metrazol-induced seizures by 6-hydroxydopamine in rat.
Exp Neurol 45:118 –133.
Dalton JC, Roberts DC and McIntyre DC (1985) Supersensitivity to the anticonvul-
sant and proconvulsant activity of clonidine following noradrenaline depletion
induced by 6-hydroxydopamine. Psychopharmacology 85:319 –322.
Ferraro G, Sardo P, Sabatino M and La Grutta V (1994) Locus coeruleus noradren-
aline system and focal penicillin hippocampal epilepsy: neurophysiological study.
Epilepsy Res 19:215–220.
Fletcher A and Forster EA (1988) A proconvulsant action of selective alpha 2-adre-
noceptor antagonists. Eur J Pharmacol 151:27–34.
Gellman RL, Kallianos JA and McNamara JO (1987) Alpha-2 receptors mediate an
endogenous noradrenergic suppression of kindling development. J Pharmacol Exp
Ther 241:891– 898.
Gobert A, Rivet JM, Audinot V, Newman-Tancredi A, Cistarelli L and Millan MJ
(1998) Simultaneous quantification of serotonin, dopamine and noradrenaline
levels in single frontal cortex dialysates of freely-moving rats reveals a complex
pattern of reciprocal auto- and heteroreceptor-mediated control of release. Neuro-
science 84:413– 429.
Gundlach AL, Burazin TC, Jenkins TA and Berkovic SF (1995) Spatiotemporal
alterations of central alpha 1-adrenergic receptor binding sites following amygda-
loid kindling seizures in the rat: autoradiographic studies using [3H]prazosin.
Brain Res 672:214 –227.
Jackson HC, Dickinson SL and Nutt DJ (1991) Exploring the pharmacology of the
pro-convulsant effects of alpha 2-adrenoceptor antagonists in mice. Psychophar-
macology 105:558 –562.
Jazrawi SP and Horton RW (1986) Brain adrenoceptor binding sites in mice suscep-
tible (DBA/2J) and resistant (C57 Bl/6) to audiogenic seizures. J Neurochem
47:173–177.
Jorm CM and Stamford JA (1993) Actions of the hypnotic anaesthetic, dexmedeto-
midine, on noradrenaline release and cell firing in rat locus coeruleus slices. Br J
Anaesth 71:447– 449.
Kirchberger K, Schmitt H, Hummel C, Peinemann A, Pauli E, Kettenmann B and
Stefan H (1998) Clonidine- and methohexital-induced epileptiform discharges
detected by magnetoencephalography (MEG) in patients with localization-related
epilepsies. Epilepsia 39:1104 –1112.
Krahl SE, Clark KB, Smith DC and Browning RA (1998) Locus coeruleus lesions
suppress the seizure-attenuating effects of vagus nerve stimulation. Epilepsia
39:709 –714.
Lazarova M and Samanin R (1983) Potentiation by yohimbine of pentylenetetrazol-
induced seizures in rats: role of alpha 2 adrenergic receptors. Pharmacol Res
Commun 15:419 – 425.
L’Heureux R, Dennis T, Curet O and Scatton B (1986) Measurement of endogenous
noradrenaline release in the rat cerebral cortex in vivo by transcortical dialysis:
effects of drugs affecting noradrenergic transmission. J Neurochem 46:1794 –1801.
Lints CE and Nyquist-Battie C (1985) A possible role for beta-adrenergic receptors in
the expression of audiogenic seizures. Pharmacol Biochem Behav 22:711–716.
Löscher W and Czuczwar SJ (1987) Comparison of drugs with different selectivity for
central alpha 1-and alpha 2-adrenoceptors in animal models of epilepsy. Epilepsy
Res 1:165–172.
Micheletti G, Warter JM, Marescaux C, Depaulis A, Tranchant C, Rumbach L and
Vergnes M (1987) Effects of drugs affecting noradrenergic neurotransmission in
rats with spontaneous petit mal-like seizures. Eur J Pharmacol 135:397– 402.
Minneman KP, Wolfe BB and Molinoff PB (1982) Selective changes in the density of
beta 1-adrenergic receptors in rat striatum following chronic drug treatment and
adrenalectomy. Brain Res 252:309 –314.
1048 Weinshenker et al.
Molinoff PB (1984) Alpha- and beta-adrenergic receptor subtypes properties, distri-
bution and regulation. Drugs 28 (Suppl 2):1–15.
Murugaiah KD and O’Donnell JM (1995) Beta adrenergic receptors facilitate nor-
epinephrine release from rat hypothalamic and hippocampal slices. Res Commun
Mol Pathol Pharmacol 90:179 –190.
Neuman RS (1986) Suppression of penicillin-induced focal epileptiform activity by
locus ceruleus stimulation: mediation by an alpha 1-adrenoceptor. Epilepsia 27:
359 –366.
Nicoletti F, Barbaccia ML, Iadarola MJ, Pozzi O and Laird HE (1986) Abnormality
of alpha 1-adrenergic receptors in the frontal cortex of epileptic rats. J Neurochem
46:270 –273.
Oishi R, Suenaga N, Hidaka T and Fukuda T (1979) The role of alpha-adrenoceptors
in the regulation of pentylenetetrazol convulsions in mice. J Pharm Pharmacol
31:709 –710.
O’Donnell JM (1990) Behavioral effects of beta adrenergic agonists and antidepres-
sant drugs after down-regulation of beta-2 adrenergic receptors by clenbuterol.
J Pharmacol Exp Ther 254:147–157.
Pandey SC, Ren X, Sagen J and Pandey GN (1995) Beta-adrenergic receptor sub-
types in stress-induced behavioral depression. Pharmacol Biochem Behav 51:339 –
344.
Papanicolaou J, Summers RJ, Vajda FJ and Louis WJ (1982) The relationship
between alpha 2-adrenoceptor selectivity and anticonvulsant effect in a series of
clonidine-like drugs. Brain Res 241:393–397.
Scotti de Carolis A, Passarelli F and Pezzola A (1986) Study on the anticonvulsant
activity of clonidine against pentylenetetrazol-induced seizures in rats: pharma-
cological evidence of alpha 2-adrenoceptors mediation. Arch Int Pharmacodyn
Ther 282:209 –218.
Szot P, Weinshenker D, White SS, Robbins CA, Rust NC, Schwartzkroin PA and
Palmiter RD (1999) Norepinephrine-deficient mice have increased susceptibility to
seizure-inducing stimuli. J Neurosci 19:10985–10992.
Thomas SA, Marck BT, Palmiter RD and Matsumoto AM (1998) Restoration of
norepinephrine and reversal of phenotypes in mice lacking dopamine beta-
hydroxylase. J Neurochem 70:2468 –2476.
Tsuda H, Ito M, Oguro K, Mutoh K, Shiraishi H, Shirasaka Y and Mikawa H (1990)
Involvement of the noradrenergic system in the seizures of epileptic El mice. Eur
J Pharmacol 176:321–330.
Waller SB and Buterbaugh GG (1985) Convulsive thresholds and severity and the
anticonvulsant effect of phenobarbital and phenytoin in adult rats administered
6-hydroxydopamine or 5,7-dihydroxytryptamine during postnatal development.
Pharmacol Biochem Behav 23:473– 478.
Weiss GK, Lewis J and Corcoran ME (1990) Antikindling effect of LC stimulation:
mediation by ascending noradrenergic projections. Exp Neurol 108:136 –140.
Wu HQ, Tullii M, Samanin R and Vezzani A (1987) Norepinephrine modulates
seizures induced by quinolinic acid in rats: selective and distinct roles of alpha-
adrenoceptor subtypes. Eur J Pharmacol 38:309 –318.
Yan QS, Dailey JW, Steenbergen JL and Jobe PC (1998) Anticonvulsant effect of
enhancement of noradrenergic transmission in the superior colliculus in geneti-
cally epilepsy-prone rats (GEPRs): a microinjection study. Brain Res 780:199 –209.
Address correspondence to: Dr. David Weinshenker, Howard Hughes Med-
ical Institute, Box 357370, University of Washington, Seattle, WA 98195.
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E-mail: dzw@genetics.washington.edu