Neuroscience 126 (2004) 795– 803
THE ANTICONVULSANT AND PROCONVULSANT EFFECTS OF ␣2-
ADRENORECEPTOR AGONISTS ARE MEDIATED BY DISTINCT
POPULATIONS OF ␣2A-ADRENORECEPTORS
P. SZOT,a,c* M. LESTER,a M. L. LAUGHLIN,b
R. D. PALMITER,b L. C. LILESd AND
D. WEINSHENKERb,d
a
Department of Psychiatry and Behavioral Sciences, University of
Washington, Seattle, WA, USA
b
Howard Hughes Medical Institute, University of Washington, Seattle,
WA, USA
c
Mental Illness Research, Education, Clinical Center, Puget Sound
Health Care System, 1660 South Columbian Way, Seattle, WA 98108,
USA
d
Department of Human Genetics, Emory University School of Medi-
cine, Atlanta, GA, USA
Abstract—The ␣2-adrenoreceptor (AR) is the most investi-
gated noradrenergic receptor with regard to modulation of
seizure activity. However, because of the complexity of mul-
tiple ␣2-AR subtypes and their distribution, the exact role of
this receptor in modulating seizure activity is not clear. ␣2A-
and ␣2C-ARs function as both autoreceptors (presynaptic) on
noradrenergic neurons, where they regulate norepinephrine
(NE) release, and as postsynaptic receptors on neurons that
receive noradrenergic innervation, where they regulate the
release of other neurotransmitters (heteroreceptor). The non-
selective ␣2-AR agonist clonidine produced a proconvulsant
effect on seizure susceptibility, while the selective ␣2A-AR
agonist guanfacine was anticonvulsant. The effects of both
␣2-AR agonists were absent in ␣2a-knockout mice, suggest-
ing that the ␣2A-AR mediates the proconvulsant and anticon-
vulsant effect of ␣2-AR agonists on seizure susceptibility. To
determine whether the ␣2-AR agonists were acting on inhib-
itory presynaptic autoreceptors to decrease NE release or on
postsynaptic receptors on NE target neurons, the effects of
clonidine and guanfacine were determined in dopamine ␤-
hydroxylase knockout (Dbh ⴚ/ⴚ) mice that lack NE. The an-
ticonvulsant effect of guanfacine persisted in Dbh ⴚ/ⴚ mice,
suggesting that guanfacine may act preferentially on ␣2A-
postsynaptic receptors that regulate the action of NE on
target neurons. In contrast, the proconvulsant effect of
clonidine was lost in Dbh ⴚ/ⴚ mice, suggesting that
clonidine may act on presynaptic autoreceptors to decrease
NE release. We hypothesize that the ␣2A-presynaptic autore-
ceptor is responsible for the proconvulsant effect of ␣2-AR
*Correspondence to: P. Szot, Mental Illness Research, Education,
Clinical Center, Puget Sound Health Care System, 1660 South Co-
lumbian Way, Seattle, WA 98108. Tel: ⫹1-206-764-2676; fax:
⫹1-206-764-2569.
E-mail address: szot@u.washington.edu (P. Szot).
Abbreviations: AR, adrenoreceptor; C56BL/6J, wild-type mouse for
␣2-adrenoreceptor knockout mouse; CT, clonic/tonic seizure; Dbh
⫺/⫺, dopamine ␤-hydroxylase knockout mouse; Dbh ⫹/⫺, dopamine
␤-hydroxylase heterozygous knockout mouse; DOPS,
L-threo-3,4-dihydroxyphenylserine; LC, locus coeruleus; NE,
norepinephrine; PTZ, pentylenetetrazol; WT, wild type; ␣2A, ␣2 sub-
type A receptor; ␣2a-AR KO, ␣2 subtype A receptor knockout mouse;
␣2B, ␣2 subtype B receptor.
0306-4522/04$30.00⫹0.00 Published by Elsevier Ltd on behalf of IBRO.
doi:10.1016/j.neuroscience.2004.04.030
795
agonists, while the ␣2A-postsynaptic receptor is responsible
for the anticonvulsant effect of ␣2-AR agonists. These data
help to clarify the inconsistent effects of ␣2-AR agonists on
seizure activity. Published by Elsevier Ltd on behalf of IBRO.
Key words: flurothyl, pentylenetetrazol, adrenergic
receptors, guanfacine, knockout mice.
There is a vast amount of literature indicating that norepi-
nephrine (NE) modulates seizure activity; however, it is
unclear how NE produces this effect. When endogenous
NE levels are reduced, by lesioning noradrenergic neurons
or genetically via targeted disruption of the dopamine ␤-
hydroxylase gene (Dbh ⫺/⫺ mice), there is an increase in
susceptibility to seizures (Mason and Corcoran, 1979;
Trottier et al., 1988; Sullivan and Osorio, 1991; Mishra et
al., 1994; Szot et al., 1999; Weinshenker et al., 2001b).
Conversely, stimulation of the locus coeruleus (LC; the
major concentration of noradrenergic cell bodies in the
CNS) inhibits seizure activity (Libet et al., 1977; Weiss et
al., 1990). Noradrenergic receptors are widely distributed
throughout the brain and are found in regions implicated in
regulating seizures such as the hippocampus, cortex and
amygdala. These data indicate that a loss of endogenous
NE is proconvulsant; however, it is unclear which adreno-
receptors (ARs) are responsible for the inhibition induced
by endogenous NE.
Conflicting results have emerged as to which ARs are
capable of modulating seizure. Each of the three different
central ARs (␣1, ␣2 and ␤) has demonstrated an anticon-
vulsant response against different convulsant agents (re-
view, Weinshenker and Szot, 2002). Interpretation of
which of the ARs are capable of modulating seizures is
further complicated by the multiple subtypes for each of the
␣-ARs. The most complex receptor is the ␣2-AR, which is
composed of three different subtypes: ␣2A-, ␣2B- and ␣2C-
AR. ␣2A- and ␣2C-ARs are localized on dendrites and
terminals of noradrenergic neurons and act as presynaptic
autoreceptors to regulate the release of NE (L’Heureux et
al., 1986; Van Gaalen et al., 1997; Kawahara et al., 1999),
as well as postsynaptic receptors on dendrites and termi-
nals of NE target cells that regulate the release of other
neurotransmitters (heteroreceptors). ␣2A-ARs are the most
abundant ␣2-AR subtype in the brain, comprising approx-
imately 90% of all central ␣2-ARs (Bucheler et al., 2002).
The highest concentration of ␣2C-AR is in the striatum,
while the ␣2B-ARs have a very limited level of expression
in the brain and apparently are not involved in regulating
seizure activity (Zeng and Lynch, 1991; Nicholas et al.,
796 P. Szot et al. / Neuroscience 126 (2004) 795– 803
1993). The ␣2-AR agonist clonidine does not discriminate
between the subtypes and has been shown to exert an
anticonvulsant, proconvulsant or no effect on seizure ac-
tivity (Oishi et al., 1979; Horton et al., 1980; Kulkarni, 1981;
Papanicolaou et al., 1982; Baran et al., 1985; Gellman et
al., 1987; Loscher and Czuczwar, 1987; Wu et al., 1987;
McIntyre and Giugno, 1988; Jackson et al., 1991; Or-
mandy et al., 1991; Amabeoku and Chandomba, 1994;
Weinshenker et al., 2001b; Weinshenker and Szot, 2002).
The ␣2-AR agonist guanfacine appears to be more selec-
tive for the ␣2A-AR (Uhlen and Wikberg, 1991; Uhlen et al.,
1992); however, there are very few studies examining the
ability of guanfacine to modulate seizure activity. Guan-
facine exerted an anticonvulsant effect against pentyle-
netetrazol (PTZ)-induced seizures (Papanicolaou et al.,
1982), a seizure model where clonidine has produced an
anti- as well as proconvulsant response (review, Wein-
shenker and Szot, 2002). Therefore, the literature supports
the ability of the noradrenergic nervous system to regulate
seizure activity, especially through the ␣2-AR. However,
because of the complexity in subtype and distribution of
␣2-ARs, the effects of ␣2-AR agonists are not always clear.
To determine which subtype of the ␣2-AR modulates
seizure activity, the ability of clonidine and guanfacine to
modulate seizure susceptibility was measured in geneti-
cally engineered mice which lack the ␣2A-AR subtype. To
determine if the conflicting data with ␣2-AR agonists is due
to differential stimulation of either presynaptic autorecep-
tors or postsynaptic receptors, we examined the effects of
clonidine and guanfacine in mice which lack endogenous
levels of NE. In mice without NE (Dbh ⫺/⫺ mice), ␣2-AR
agonists cannot produce their effects through the presyn-
aptic autoreceptor, because the main function of this re-
ceptor is to modulate the release of NE (L’Heureux et al.,
1986; Van Gaalen et al., 1997; Kawahara et al., 1999).
Therefore, any effect an ␣2-AR agonist produces in the
Dbh ⫺/⫺ mouse indicates a non-autoreceptor-mediated
effect (i.e. postsynaptic). These studies were designed to
determine the importance of the ␣2-AR in regulating sei-
zure activity and potentially indicate the value of guan-
facine as a new pharmacological agent for the treatment of
epilepsy.
EXPERIMENTAL PROCEDURES
Dbh knockout mice
Dbh ⫺/⫺ mice, maintained on a mixed 129/SvEv and C57BL6/J
hybrid background, were generated as previously described
(Thomas et al., 1995). Mice were reared in a specific pathogen-
free facility with a 12-h light/dark cycle at the University of Wash-
ington (Seattle, WA, USA), and moved to a conventional facility for
seizure experiments. Mice between 3 and 6 months of age were
used for all experiments. 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 Washing-
ton Animal Care Committee. The minimum number of animals
were used for these studies and care was taken to minimize any
suffering. Genotype was deduced from phenotype, Dbh ⫺/⫺ mice
exhibit delayed growth during adolescence and ptosis (Thomas et
al., 1995). A subset of mice was confirmed by PCR. Dbh ⫾ mice,
which were used as controls, are indistinguishable from wild-type
(WT; ⫹/⫹) mice as to NE and epinephrine levels (Thomas et al.,
1998) and for previously tested behaviors, including seizure sus-
ceptibility (Thomas et al., 1998; Szot et al., 1999).
␣2a- and ␣2c-AR knockout mice (␣2a-AR KO)
␣2a-AR KO and ␣2c-AR KO mice, maintained on a pure C57BL6/J
background, were generated as previously described (Link et al.,
1995, 1996; MacMillan et al., 1996). ␣2c ⫹/⫺ and ␣2ac ⫹/⫺ mice
were obtained from Brian K. Kobilka (Stanford University, Stan-
ford, CA, USA); these animals were used to generate WT
(C57BL6/J; ␣2a/␣2c; ⫹/⫹/⫹/⫹), ␣2a⫺/⫺ (␣2a/␣2c; ⫺/⫺/⫹/⫹) and
␣2c⫺/⫺ (␣2a/␣2c; ⫹/⫹/⫺/⫺) KO mice. Genotypes were determined
by PCR. The ␣2a- and ␣2c-AR KO mice were housed in the same
facility as the Dbh ⫹/⫺ and Dbh ⫺/⫺ mice; thus, all sets of
animals experienced identical environmental conditions.
Flurothyl seizure induction
Mice were placed in an air-tight Plexiglas chamber, and the vol-
atile convulsant flurothyl (Aldrich, Milwaukee, WI, USA) was in-
fused (20 ␮l/min) onto filter paper from which it vaporized (Szot et
al., 1999). The latency (seconds) to the first generalized clonic/
tonic (CT) seizure served as the measurement of seizure suscep-
tibility. Each animal was tested individually, removed immediately
from the chamber after seizure onset, and received only one
exposure to flurothyl. Also recorded were the number of animals
(genotypes and drug treatments) that progressed to tonic exten-
sion once the animals were removed from the chamber. An in-
crease in the percentage of animals that progress to tonic exten-
sion indicates an increase in seizure severity (i.e. increased neu-
ronal activity).
PTZ seizure-induction
PTZ (Sigma, St. Louis, MO, USA) seizure-induction was per-
formed as previously described (Weinshenker et al., 2001b). Be-
cause Dbh ⫺/⫺ mice are more sensitive to PTZ-induced seizures
(Szot et al., 1999), doses of PTZ were adjusted based on geno-
type to elicit seizures of similar severity in Dbh ⫹/⫺ and Dbh ⫺/⫺
mice. PTZ was administered at a dose of 50 mg/kg, i.p. (12.5
mg/ml, 4 ml/kg) to Dbh ⫹/⫺ mice and 40 mg/kg, i.p. (10 mg/kg,
4 ml/kg) to Dbh ⫺/⫺ mice. PTZ was administered at a dose of
40 mg/kg, i.p. (10 mg/kg, 4 ml/kg) to the WT C57BL6/J and ␣2a-AR
KO mice. All mice were placed in a clear Plexiglas chamber and
closely monitored for 10 min. This observation time was chosen
because mice that displayed seizure activity did so within the first
few minutes after PTZ administration. Latency to the first CT
seizure was recorded. Animals that did not experience this seizure
behavior were assigned a latency of 600 s for that measurement.
Pharmacological agents
All drugs were dissolved in normal saline. To test the effect of
␣2-AR agonists, clonidine (␣2-AR) and guanfacine (␣2A-AR;
Sigma) were administered i.p. in a volume of 4 ml/kg, 30 min prior
to seizure induction. In Dbh ⫹/⫺ and Dbh ⫺/⫺ mice the following
AR agonists were also administered in a volume of 4 ml/kg 30 min
prior to flurothyl seizure testing: cirazoline (␣1-AR; 0.2 mg/kg, i.p.),
dobutamine (␤1-AR; 10 mg/kg, i.p.) and albuterol (␤2-AR; 10 mg/
kg, i.p.). These doses have been used previously in rodents with
other convulsant agents (Weinshenker et al., 2001b; Weinshenker
and Szot, 2002) and against PTZ-induced seizures where cirazo-
line and albuterol demonstrated an anticonvulsant effect in Dbh
⫺/⫺ mice (Weinshenker et al., 2001b). When agonists were com-
bined, they were made as a “cocktail” and given as a single
injection at a volume of 4 ml/kg 30 min before seizure testing. A
subset of Dbh ⫺/⫺ mice received a single i.p. injection of L-threo-
 P. Szot et al. / Neuroscience 126 (2004) 795– 803 797

A B
600
saline saline
CT Seizure Latency (sec) 500
clonidine *** guanfacine

400
Flurothyl

300

***
200
***
100

0
WT α2c -KO α2a-KO WT α2a-KO
Fig. 1. Effects of clonidine (A) and guanfacine (B) on flurothyl-induced seizures in WT, ␣2a-AR KO and ␣2c-AR KO mice. Shown are latencies
(mean⫾S.E.M.) to CT seizure (seconds). (A) Saline (WT C57BL6/J, n⫽7; ␣2c-AR KO, n⫽9; ␣2a-AR KO, n⫽4) or clonidine (0.1 mg/kg, i.p.; WT
C57BL6/J, n⫽7; ␣2c-AR KO, n⫽10; ␣2a-AR KO, n⫽4) administered 30 min prior to flurothyl. (B) Saline (WT C57BL6/J, n⫽10; ␣2a-AR KO, n⫽8) or
guanfacine (0.1 mg/kg, i.p.; WT C57BL6/J, n⫽7; ␣2a-AR KO, n⫽8) was administered 30 min prior to flurothyl. *** Indicates a significant difference of
treatment group from its respective control, P⬍0.001.

3,4-dihydroxyphenylserine (DOPS; 1 mg/kg⫹2 mg/ml vitamin C)
6 h prior to flurothyl seizure testing. DOPS is converted to NE by
aromatic L-amino acid decarboxylase, which is present in all bio-
genic 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).
Statistics
Latency (seconds) to CT seizure for flurothyl and PTZ data from
each group on a given test day between drug- and saline-treated
animals were expressed as the mean⫾S.E.M. and were analyzed
with Student’s t-test comparisons using Stat View program; differ-
ences were considered significant when P⬍0.05. Kruskal-Wallis
test followed by Dunn’s multiple comparison post hoc test was
used for comparing means from more than two groups; statistical
significance was taken at P⬍0.05. An anticonvulsant response is
observed as an increase in the latency to CT seizure, while a
proconvulsant response produces a decrease in the latency to CT
seizure.
RESULTS
Effect of clonidine and guanfacine on flurothyl-
induce seizures in ␣2A-AR KO mice
Seizure latencies for saline-treated WT C57Bl6/J and
␣2a-AR KO mice were similar, indicating that the loss of the
␣2A-AR did not affect flurothyl seizure susceptibility (Fig.
1). However, the loss of the ␣2A-AR resulted in more
severe seizures; all (100%) of the ␣2a-AR KO mice which
received saline progressed to tonic extension with flurothyl
seizure-induction, while none (0%) of the WT C57Bl6/J or
␣2c-AR KO mice which received saline progressed to tonic
extension. The administration of either ␣2-AR agonist
(clonidine or guanfacine) did not modify the enhanced
seizure severity of the ␣2a-AR KO mice to flurothyl.
Administration of clonidine (0.1 mg/kg, i.p.) to WT
C57BL6/J mice resulted in a proconvulsant response to
flurothyl (Fig. 1A). To determine which of the ␣2-AR sub-
types mediates the proconvulsant response of clonidine,
flurothyl-induced seizures were measured in ␣2a- and
␣2c-AR KO mice 30 min after the administration of either
saline or clonidine. The proconvulsant effect of clonidine
was absent only in ␣2a-AR KO mice but persisted in
␣2c-AR KO (Fig. 1 A), demonstrating that the ␣2A-AR me-
diates this effect.
Guanfacine (0.1 mg/kg), in contrast to clonidine, was
anticonvulsant in WT C57Bl6/J mice (Fig. 1B). This an-
ticonvulsant response of guanfacine is also due to stim-
ulation of the ␣2A-AR, as the anticonvulsant response of
guanfacine is absent in ␣2a-AR KO mice (Fig. 1B).
These data indicate that the proconvulsant action of
clonidine and the anticonvulsant action of guanfacine
against flurothyl-induced seizures are mediated by the
␣2A-AR subtype.
Effect of guanfacine on PTZ-induce seizures in
␣2a-AR KO mice
In contrast to flurothyl-induced seizures, the loss of the
␣2A-AR resulted in an animal less susceptible to PTZ-
induced seizures; saline treated ␣2a-AR KO mice had sig-
nificantly longer latency than WT C57Bl6/J mice adminis-
tered saline (Fig. 2). However, as with flurothyl, all (100%)
of ␣2a-AR KO mice that exhibited CT seizures progressed
to tonic extension, while none (0%) of the WT C57Bl6/J
798 P. Szot et al. / Neuroscience 126 (2004) 795– 803

Effect of clonidine and guanfacine against flurothyl-

CT Seizure Latency (sec) saline induced seizures in Dbh ⴙ/ⴚ and Dbh ⴚ/ⴚ mice
600 guanfacine † Clonidine (0.1 mg/kg, i.p.) produced a proconvulsant re-
sponse against flurothyl-induced seizures in Dbh ⫹/⫺
500 mice (Fig. 3A). Dbh ⫹/⫺ mice are indistinguishable from

400
* their WT mice as to NE and epinephrine levels (Thomas et
al., 1998) and for previously tested behaviors, including
PTZ

seizure susceptibility (Thomas et al., 1998; Szot et al.,

300
200
100
0
WT α 2a-KO
Fig. 2. Effect guanfacine on PTZ-induced seizures in WT and ␣2a-AR
KO. Shown are latencies (mean⫾S.E.M.) to CT seizure (seconds) for
saline (WT C57BL6/J, n⫽5; ␣2a-AR KO, n⫽6) or guanfacine (0.1 mg/
kg, i.p.; WT C57BL6/J, n⫽6; ␣2a-AR KO, n⫽6) administered 30 min
prior to PTZ. * Indicates a significant difference of treatment group
from its respective control, P⬍0.05. † Indicates a significant difference
of saline ␣2a-AR KO from saline WT C57BL6/J, P⬍0.01.
mice progressed to tonic extension with PTZ. As with
flurothyl-induced seizures, guanfacine produced an anti-
convulsant effect against PTZ-induced seizures in the WT
C57Bl6/J, but not ␣2a-AR KO mice (Fig. 2), indicating the
␣2A-AR was mediating this effect.
1999). Guanfacine at a dose of 0.1 mg/kg did not affect
flurothyl seizure susceptibility, but a higher dose of guan-
facine (1.0 mg/kg, i.p.) produced a significant anticonvul-
sant response in Dbh ⫹/⫺ mice to flurothyl (Fig. 3B).
These responses of Dbh ⫹/⫺ mice to clonidine and guan-
facine are similar to those observed in WT C57Bl6/J mice
(Fig. 1).
To determine if other ARs may modulate flurothyl-in-
duced seizures, an ␣1-AR agonist (cirazoline), a ␤1-AR ago-
nist (dobutamine) and a ␤2-AR agonist (albuterol) were ad-
ministered to Dbh ⫹/⫺ mice and flurothyl seizure suscepti-
bility was tested. None of these other AR agonists affected
flurothyl-induced seizures in Dbh ⫹/⫺ mice (Fig. 3C).
As previously reported Dbh ⫺/⫺ mice have signifi-
cantly shorter latency to flurothyl-induced seizures than
Dbh ⫹/⫺ mice (Fig. 4A; histogram bar compared with
dashed line which represents latency of Dbh ⫹/⫺ mice
administered saline). Also as previously indicated (Szot et
al., 1999), a greater number of Dbh ⫺/⫺ mice progressed
to tonic extension following flurothyl-induced seizures than
Dbh ⫹/⫺ mice administered saline (data not shown), indi-
cating the loss of endogenous NE results in an animal
more susceptible to flurothyl-induced seizures.

140 A B C
% Change from Dbh +/-mice

120 *
100
*
treated with saline

80
60
40
20
0 0.1 1.0
Clon Cir Dob Alb
guanfacine
(mg/kg)
Fig. 3. Effects of AR-agonists on flurothyl-induced seizure susceptibility in Dbh ⫹/⫺ mice. Shown are the percent (%) changes (mean⫾S.E.M.) in CT
seizure threshold of Dbh ⫹/⫺ mice administered either: (A) clonidine (Clon; ␣2-agonist, n⫽9), (B) guanfacine (Guan; ␣2A-agonist) 0.1 mg/kg (n⫽4)
and Guan 1.0 mg/kg (n⫽6); or (C) cirazoline (Cir; ␣1-agonist, n⫽12), dobutamine (Dob; ␤1-agonist, n⫽6), or albuterol (Alb; ␤2-agonist, n⫽7. To obtain
% change in CT threshold, CT threshold values (seconds) of Dbh ⫹/⫺ mice given drug treatment was compared with its respective group of saline
Dbh ⫹/⫺ mice on a given day that seizures were tested. The dashed line represents the saline Dbh ⫹/⫺ mice CT threshold (100%). A histogram bar
crossing the dashed line indicates a drug treatments ability to produce an anticonvulsant response in Dbh ⫹/⫺ mice. * Indicates a significant difference
of treatment group from its respective control group that was obtained on a single day of testing, P⬍0.05.
 P. Szot et al. / Neuroscience 126 (2004) 795– 803 799

180 A B C D E F
% Change from Dbh -/- 160 * * *
140 *
120 †
100
80
60
40
20
0 saline Clon Guan Cir Dob Alb DOPS Clonidine + + + + Guan
Cirazoline + + +
Dob
Dobutamine + + + + + +
Albuterol + + + + + Alb

Fig. 4. Effects of AR-agonists on flurothyl-induced seizure susceptibility in Dbh ⫺/⫺ mice. Shown are the percent (%) changes (mean⫾S.E.M.) in CT
seizure threshold of Dbh ⫺/⫺ mice administered either: (A) saline (Dbh ⫺/⫺ mice, n⫽68); (B) clonidine (Clon; ␣2-agonist, n⫽10) or guanfacine (Guan;
␣2A-agonist, n⫽5); (C) cirazoline (Cir; ␣1-agonist, n⫽7), dobutamine (Dob; ␤1-agonist, n⫽8), or albuterol (Alb; ␤2-agonist, n⫽7); (D) DOPS (n⫽7); (E)
combination of AR agonists (Cir/Clon/Dob/Alb, n⫽7; Cir/Dob/Alb, n⫽6; Clon/Dob/Alb, n⫽6; Dob/Alb, n⫽6; Clon/Dob, n⫽4, and Clon/Alb, n⫽5); or (F)
combination of Guan/Dob/Alb (n⫽6). To obtain % change in CT threshold, CT threshold values (seconds) of Dbh ⫺/⫺ mice given drug treatment were
compared with their respective group of Dbh ⫺/⫺ mice administered saline on a given day that seizures were tested. The dashed line represents the
% difference of Dbh ⫹/⫺ mice CT threshold from saline Dbh ⫺/⫺ mice. A histogram bar crossing the dashed line indicates a drug treatments ability
to normalize the seizure susceptibility of the Dbh ⫺/⫺ mice to that observed in saline Dbh ⫹/⫺ mice. * Indicates a significant difference of treatment
group from its respective control group that was obtained on a single day of testing, P⬍0.05. † Indicates a significant difference in flurothyl seizure
latency of Dbh ⫺/⫺ mice administered saline as compared with Dbh ⫹/⫺ mice administered saline (dashed line), P⬍0.05.

Administration of either clonidine (0.1 mg/kg) or guan-
facine (1 mg/kg) alone to Dbh ⫺/⫺ mice did not signifi-
cantly affect seizure susceptibility to flurothyl (Fig. 4B).
However, administration of clonidine or guanfacine re-
duced the severity of flurothyl-induced seizures. Clonidine
reduced the number of Dbh ⫺/⫺ mice that progressed to
tonic extension from 70% (saline) to 55%; while guan-
facine reduced the number of animals that progressed to
tonic extension from 60% (saline) to 40%.
Since ␣1- and ␤2-AR agonists produced an anticonvul-
sant effect in Dbh ⫺/⫺ mice against PTZ-induced seizures
(Weinshenker et al., 2001b), we administered ␣1- (cirazo-
line), ␤1- (dobutamine) and ␤2-AR (albuterol), and non-
selective ␤-AR (isoproterenol, 10 mg/kg) agonists in doses
similar to those used with PTZ (Weinshenker et al.,
2001b). Single administration of these AR agonists did not
significantly affect seizure susceptibility in Dbh ⫺/⫺ mice
(Fig. 4C and data not shown).
To reproduce the actions of endogenous NE in Dbh
⫺/⫺ mice, two experiments were performed: 1) adminis-
tration of DOPS to elevate endogenous levels of NE
and 2) a “cocktail” containing all the AR agonists
(clonidine⫹cirazoline⫹dobutamine⫹albuterol) was given
and flurothyl seizure testing was performed. Administration
of DOPS, as previously shown (Szot et al., 1999) normal-
ized Dbh ⫺/⫺ mice flurothyl seizure threshold to that of
Dbh ⫹/⫺ mice (Fig. 4D). The AR agonist cocktail also
produced an anticonvulsant effect in Dbh ⫺/⫺ mice (Fig.
4E). To determine which AR agonists were responsible for
the anticonvulsant effect of this cocktail, a single AR ago-
nist was removed and then the new “cocktail” was re-
tested. Removal of cirazoline (cocktail consists of
clonidine⫹dobutamine⫹albuterol) still resulted in an anti-
convulsant effect in Dbh ⫺/⫺ mice (Fig. 4E). However,
when ␣2-AR agonist clonidine was removed, leaving only
dobutamine⫹albuterol, the anticonvulsant response was
lost (Fig. 4E). The combination of cirazoline with the ␤-AR
agonists was not anticonvulsant (Fig. 4E). The combina-
tion of clonidine with either dobutamine or albuterol did not
result in a significant anticonvulsant response in Dbh ⫺/⫺
mice (Fig. 4E). Replacing clonidine with guanfacine
(1.0 mg/kg, i.p.) with ␤1- and ␤2-AR agonists also resulted
in an anticonvulsant effect (Fig. 4F). Therefore, the com-
bination of an ␣2-AR agonist with ␤-AR agonists is required
to normalize flurothyl-seizure susceptibility in mice that
lack endogenous NE.
Effect of guanfacine against PTZ-induced seizures in
Dbh ⴙ/ⴚ and Dbh ⴚ/ⴚ mice
Previous work examined the ability of different noradren-
ergic agonists and antagonists to modify PTZ-induced sei-
zures in Dbh ⫹/⫺ and Dbh ⫺/⫺ mice (Weinshenker et al.,
2001b); however guanfacine was not tested. Guanfacine
demonstrated a dose dependent anticonvulsant effect in
Dbh ⫹/⫺ mice against PTZ-induced seizures with the
1.0 mg/kg dose demonstrating a significant difference from
saline-treated animals (Fig. 5). This is very similar to the
effects of guanfacine against flurothyl-induced seizures in
Dbh ⫹/⫺ mice. However, unlike flurothyl seizures, admin-
istration of guanfacine alone at either dose (0.1 or 1 mg/kg)
produced a significant anticonvulsant effect in Dbh ⫺/⫺
mice (Fig. 5). Therefore, the enhanced susceptibility of
Dbh ⫺/⫺ mice to PTZ can be prevented by the adminis-
tration of either a ␣1- or ␤2-AR agonists as previously
shown (Weinshenker et al., 2001b) and by the ␣2A-AR
agonist guanfacine.
800
CT Seziure Latency (sec)
PTZ
Fig. 5. Effect of guanfacine on PTZ-induced seizure susceptibility in Dbh ⫹/⫺ and Dbh ⫺/⫺ mice. Shown are latencies (mean⫹/⫺S.E.M.) to CT
seizure (seconds) for saline (Dbh ⫹/⫺, n⫽8; Dbh ⫺/⫺, n⫽9) or guanfacine 0.1 mg/kg, i.p. (Dbh ⫹/⫺, n⫽8; Dbh ⫺/⫺, n⫽7) or guanfacine 1 mg/kg,
i.p. (Dbh ⫹/⫺, n⫽8; Dbh ⫺/⫺, n⫽8) administered 30 min prior to PTZ. * Indicates a significant difference of treatment group from its respective control,
P⬍0.05.
DISCUSSION
Dbh ⴚ/ⴚ mice and ␣2a-AR KO mice have increased
seizure activity
The loss of NE or the ␣2A-AR results in an animal with
increased seizure activity, but the two different genotypes
demonstrate this increased activity differently. The Dbh
⫺/⫺ mouse has a shorter latency and more severe sei-
zures to both flurothyl- and PTZ-induced seizures than the
Dbh ⫹/⫺ mouse (Szot et al., 1999; Weinshenker et al.,
2001b). Against flurothyl-induced seizures the ␣2a-AR KO
mouse has normal latency but more severe seizures, while
against PTZ-induced seizures the loss of the ␣2A-AR re-
sults in an animal with a longer latency, but more severe
seizures. These data indicate that the loss of NE and a
particular ␣2-AR subtype can modulate both flurothyl- and
PTZ-induced seizures.
The ␣2-AR agonists clonidine and guanfacine have
different effects on flurothyl- and PTZ-induced
seizures; however both drugs mediate their effects
through the ␣2A-AR
The ␣2-AR agonists clonidine and guanfacine affected flu-
rothyl-induced seizures differently in mice which have nor-
mal levels of NE (WT C57BL6/J and Dbh ⫹/⫺ mice);
clonidine was proconvulsant, while guanfacine was anti-
convulsant. Similarly with PTZ-induced seizures, clonidine
tended to exhibit a proconvulsant effect in Dbh ⫹/⫺ mice
(Weinshenker et al., 2001b), while guanfacine was anti-
convulsant. The ␣2A-AR appeared to be responsible for
both the proconvulsant response of clonidine and the an-
ticonvulsant response of guanfacine, as both of these ef-
fects were absent in ␣2a-AR KO mice. These data indicate
that the non-selectivity of clonidine for the different ␣2-AR
subtypes (Newman-Tancredi et al., 1998) cannot explain
the opposite effects of clonidine and guanfacine on seizure
P. Szot et al. / Neuroscience 126 (2004) 795– 803
Dbh +/- mice Dbh -/- mice
500
400 * *
*
300
200
100
0
saline 0.1 1.0 saline 0.1 1.0
guanfacine guanfacine
susceptibility. The varied effects of ␣2-AR agonists against
flurothyl- and PTZ-induced seizures may reflect the ability
of these drugs to differentially activate ␣2A-ARs localized
as presynaptic autoreceptors compared with postsynaptic
receptors localized on target neurons (Zeng and Lynch,
1991; Nicholas et al., 1993; Bucheler et al., 2002).
Activation of noradrenergic ␣2A-presynaptic
autoreceptors produces a proconvulsant response,
while ␣2A-postsynaptic receptors are anticonvulsant
Mice that lack endogenous levels of NE (Dbh ⫺/⫺ mice)
were used to define if the opposite effects of clonidine and
guanfacine on seizure susceptibility were due to stimula-
tion of either presynaptic autoreceptors or postsynaptic
receptors on target neurons. The rationale is that in mice
without endogenous NE, ␣2-AR agonists cannot produce
an effect mediated through the autoreceptor and modula-
tion of NE release, since there is no endogenous NE to
modulate. In contrast, a response mediated by stimulation
of postsynaptic receptors which are found on dendrites
and terminals of NE target neurons would persist in mice
which lack NE because AR distribution and density are
normal in Dbh ⫺/⫺ mice. These experiments are possible
because unlike lesioning studies of noradrenergic neurons
that deplete endogenous NE levels, Dbh ⫺/⫺ mice have
normal levels of ␣2- and ␣1-AR binding sites and ␣2A-AR
mRNA expression in the LC (Szot, unpublished observa-
tion and C. Murrin, personal communication) as compared
with Dbh ⫹/⫺ mice. In addition, the co-transmitters that are
released with NE may not play a major role in modulating
CT seizures induced by flurothyl (Weinshenker et al.,
2001a).
Surprisingly, for flurothyl-induced seizures we found
that none of the AR agonists administered alone af-
fected seizure susceptibility of Dbh ⫺/⫺ mice. The lack
of response of Dbh ⫺/⫺ mice to AR agonists is not a
 P. Szot et al. / Neuroscience 126 (2004) 795– 803
strain background issue because guanfacine produced
an anticonvulsant effect against flurothyl-induced sei-
zures in Dbh ⫹/⫺ mice. Also restoration of endogenous
levels of NE with DOPS normalized flurothyl-induced
seizures in Dbh ⫺/⫺ mice (Fig. 4D; Szot et al., 1999),
indicating that stimulation of noradrenergic receptors
can modulate flurothyl-induced seizures in Dbh ⫺/⫺
mice. It is also apparent that agonist stimulation of ARs
is intact in Dbh ⫺/⫺ mice because ␣1- and ␤2-AR ago-
nists are anticonvulsant against PTZ-induced seizures
(Weinshenker et al., 2001b), and a cocktail of AR ago-
nists restores normal seizure susceptibility to flurothyl-
induced seizures. This anticonvulsant effect of AR ago-
nist cocktail in Dbh ⫺/⫺ mice is due to the combination
of ␣2-AR (either clonidine or guanfacine), ␤1- and ␤2-AR
agonists. It is unclear why this combination of AR ago-
nists is required to produce an anticonvulsant response
against flurothyl-induced seizures, but the ␣2-AR ap-
pears to be important in this combination of agents. We
speculate the ␣2A-postsynaptic receptors are involved in
mediating this anticonvulsant effect of ␣2-AR with ␤-AR
agonists in
Dbh ⫺/⫺ mice against flurothyl-induced seizures. Fur-
ther support for a postsynaptic ␣2-AR mediated anticon-
vulsant response is the requirement of the ␤-AR ago-
nists which are localized only postsynaptic to noradren-
ergic terminals.
The anticonvulsant action of ␣2A-postsynaptic receptor
is seen with guanfacine against PTZ-induced seizures.
Guanfacine, at a low dose of 0.1 mg/kg and a high dose of
1.0 mg/kg, produced a significant anticonvulsant effect in
Dbh ⫺/⫺ mice against PTZ-induced seizures, while under
similar conditions clonidine alone had no effect (Wein-
shenker et al., 2001b).
A difference in response between receptor subpopu-
lations differentially activated by clonidine and guan-
facine has been observed with spatial working memory.
Guanfacine produced a dose-dependent enhancement
of spatial working memory, while clonidine had no effect
or at times impaired memory (Franowicz and Arnsten,
1999; Jakala et al., 1999; Birnbaum et al., 2000). The
selectivity of guanfacine for the ␣2A-postsynaptic recep-
tor was proposed for the difference in the ability of
clonidine and guanfacine to modulate spatial working
memory,that guanfacine was selectively stimulating the ␣2A-
postsynaptic receptor in the prefrontal cortex (Tanila et al.,
1996; Avery et al., 2000; Franowicz et al., 2002), while
stimulation of the ␣2A-presynaptic autoreceptor with
clonidine disrupts memory (Jakala et al., 1999). This hy-
pothesis was supported by experiments in animals that
had NE levels depleted either by reserpine (Cai et al.,
1993; Franowicz and Arnsten, 1999) or by the administra-
tion of a noradrenergic neurotoxin (Schneider and
Kovelowski, 1990). ␣2-AR agonists continued to enhance
spatial working memory in the absence of endogenous NE,
indicating an effect being mediated through the ␣2-
postsynaptic receptor.
801
CONCLUSION
The data generated in this study indicate that the nor-
adrenergic system modulates flurothyl- and PTZ-in-
duced seizures via the ␣2-AR. The ␣2A-AR subtype ap-
pears to be the major ␣2-AR subtype involved in medi-
ating the action of both clonidine and guanfacine against
flurothyl- and PTZ-induced seizures. We speculate that
stimulation of ␣2A-postsynaptic receptors on NE target
neurons is responsible for this anticonvulsant response,
possibly suppressing the release of excitatory neuro-
transmitters (Giorgi et al., 2003) in regions such as the
hippocampus, cortex or amygdala. Future work will de-
termine the CNS region involved in the anticonvulsant
action of guanfacine. While stimulation of the presynap-
tic autoreceptor is responsible for a proconvulsant re-
sponse by reducing activity of LC neurons or the release
of NE into the synapse in regions like the hippocampus,
cortex or amygdala, which would then render the animal
more sensitive to convulsant stimuli, e.g. Dbh ⫺/⫺ mice.
It appears that guanfacine has a higher selectivity for
␣2A-postsynaptic receptor because it produces an anti-
convulsant response in animals with and without endog-
enous NE; while clonidine, under these conditions, ap-
pears to be more selective for the presynaptic autore-
ceptor because it produces a proconvulsant response.
However, clonidine can also produce an anticonvulsant
effect (Kulkarni, 1981; Papanicolaou et al., 1982; Gell-
man et al., 1987; Loscher and Czuczwar, 1987; McIntyre
and Giugno, 1988; Amabeoku and Chandomba, 1994;
unpublished observations) and when it does, we hypoth-
esize it is due to stimulation of postsynaptic receptors on
NE target neurons. In support of this hypothesis, earlier
studies have attempted to define the location of the
anticonvulsant effect of clonidine. These earlier studies
utilized animals with their noradrenergic nervous system
lesioned (Dalton et al., 1985; McIntyre and Giugno,
1988). The results from these studies concluded the
anticonvulsant effect of clonidine was due to postsynap-
tic ␣2-AR.
These studies help to clarify the variable effects of
clonidine against different convulsant agents and suggest
that guanfacine warrants further investigation as an ␣2-AR
agonist to suppress seizure activity.
Acknowledgements—We thank Sumitomo Pharmaceuticals for
the generous donation of L-threo-3,4-dihydroxyphenylserine
(DOPS) and Dr. Brian Kobilka (Stanford University) for the ␣2-AR
knockout mice. P.S. was supported by the National Alliance for
Research on Schizophrenia and Depression, Pediatric Epilepsy
Research Center (University of Washington) and the Department
of Veterans Affairs.
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(Accepted 22 April 2004)

(Available online 24 May 2004)