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Modulation of Hippocampal Excitability and Seizures by Galanin
Andrey M. Mazarati,
1,4
John G. Hohmann,
6,8
Andrea Bacon,
5
Hantao Liu,
1,4
Raman Sankar,
2,3
Robert A. Steiner,
6,7,8
David Wynick,
5
and Claude G. Wasterlain
1,3,4
1
Departments of Neurology,
2
Pediatrics, and
3
Brain Research Institute, University of California Los Angeles, School of
Medicine, Los Angeles, California 90095-1769,
4
Epilepsy Research Laboratories, Veterans Affairs, Greater Los Angeles
Healthcare System, Sepulveda, California 91343-2099,
5
Department of Medicine, Bristol University, Bristol, BS2-8HW
United Kingdom, and
6
Neurobiology and Behavior Program and Departments of
7
Obstetrics and Gynecology, and
8
Physiology and Biophysics, University of Washington, Seattle, Washington 98195-7290
Previous studies have shown that the expression of the neu-
ropeptide galanin in the hippocampus is altered by seizures and
that exogenous administration of galanin into the hippocampus
attenuates seizure severity. To address the role of endogenous
galanin in modulation of hippocampal excitability and its possible
role in seizure mechanisms, we studied two types of transgenic
mice: mice with a targeted disruption of the galanin gene (GalKO)
and mice that overexpress the galanin gene under a dopamine-

-hydroxylase promoter (GalOE). GalKO mice showed increased
propensity to develop status epilepticus after perforant path
stimulation or systemic kainic acid, as well as greater severity of
pentylenetetrazol-induced convulsions. By contrast, GalOE mice
had increased resistance to seizure induction in all three models.
Physiological tests of hippocampal excitability revealed en-
hanced perforant path–dentate gyrus long-term potentiation
(LTP) in GalKO and reduced LTP in GalOE. GalKO showed in-
creased duration of afterdischarge (AD) evoked from the dentate
gyrus by perforant path simulation, whereas GalOE had in-
creased threshold for AD induction. Depolarization-induced glu-
tamate release from hippocampal slices was greater in GalKO
and lower in GalOE, suggesting that alterations of physiological
and seizure responses in galanin transgenic animals may be
mediated through modulation of glutamate release.
Our data provide further evidence that hippocampal galanin
acts as an endogenous anticonvulsant and suggest that genet-
ically induced changes in galanin expression modulate both
hippocampal excitability and predisposition to epileptic seizures.
Key words: galanin; transgenic mice; seizures; hippocampus;
long-term potentiation; glutamate
The neuropeptide galanin is widely distributed through the brain
of various species (Skofitsch and Jacobowitz, 1985, 1986; Melander
et al., 1986; Merchenthaler et al., 1993) and is a potent neuroen-
docrine regulator of hypothalamo-adrenal hormone release, feed-
ing behavior, insulin secretion, and NPY release (Bauer et al.,
1986; Bartfai et al., 1993; Merchenthaler et al., 1993; Wynick et al.,
1998). The hippocampus contains few galanin-immunoreactive
neurons, but receives abundant galaninergic input from the medial
septum, locus coeruleus, and hypothalamus (Melander et al., 1986;
Lamour et al., 1988; Senut et al., 1989; Cortes et al., 1990; Merch-
enthaler et al., 1993). The density of galanin-containing fibers is
especially high in the dentate gyrus and CA3 (Mazarati et al.,
1998a).
Among the effects of galanin in the hippocampus, presynaptic
inhibition of release of the excitatory neurotransmitter glutamate
from principal cells, mediated through opening of ATP-dependent
K channels, is of special interest (Zini et al., 1993a,b), because it
could modulate hippocampal excitability. Indeed, several lines of
research suggest a novel role for galanin in seizures. Thus, the
seizure-induced depletion of galanin from the rat hippocampus is
associated with the development of self-sustaining status epilepti-
cus (SSSE) (Mazarati et al., 1998b); the injection of galanin into
the hippocampus attenuates seizure activity, whereas galanin an-
tagonists facilitate it (Mazarati et al., 1992, 1998a). However, the
functional significance of endogenous galanin in regulating hip-
pocampal excitability and seizures remains poorly understood.
The study of animals with genetically induced alterations in
galanin expression could help to elucidate the contribution of
galanin in the regulation of hippocampal function and lend insights
into the pathophysiology and treatment of epileptic seizures. We
examined hippocampal function and seizure susceptibility in two
lines of transgenic mice: one with a targeted deletion of the galanin
gene (Wynick et al., 1998; Kerr et al., 2000) and another that
overexpresses the galanin gene under the control of the dopamine

-hydroxylase promoter (Hohmann et al., 1997). Previous analysis
of galanin-overexpressing mice (GalOE) revealed increased gala-
nin mRNA in piriform and entorhinal cortices, subiculum, and in
noradrenergic cell groups in the brainstem (Hohmann et al., 1997).
We found significantly higher (160%) levels of galanin, as mea-
sured by ELISA, in the brainstem and entorhinal cortex of GalOE
compared to wild-type (WT) controls (H. Liu, A. Mazarati, and C.
Wasterlain, unpublished data). Studies of galanin knock-out mice
(GalKO) revealed undetectable levels of galanin in both the cortex
and hypothalamus (Wynick et al., 1998). Therefore, these two
types of animals provide a convenient tool for studying the physi-
ological and pathological roles of endogenous galanin.
We provide evidence that altered expression of the galanin gene
affects both hippocampal excitability and predisposition to sei-
zures, suggesting that endogenous galanin may play an important
role as an endogenous anticonvulsant. These findings raise the
possibility that genetic deficits in galanin expression may be a
contributory factor in certain forms of epilepsy.
MATERIALS AND METHODS
Animals. GalKO were bred on the pure 129OlaHsd strain for at least 10
generations. Development of GalKO has been described in detail earlier
(Wynick et al., 1998; Kerr et al., 2000). In brief, the entire coding region of
the galanin gene was deleted. The generation of GalKO was performed
Received March 10, 2000; revised June 2, 2000; accepted June 5, 2000.
This work was supported by Grants NS 11315 from National Institutes of Health and
by the Research Service of Veteran Health Administration (C.W.), NS01792 from
National Institutes of Health (R.S.), RO1-HD27142 and U54-HD12626 from the
National Institute of Child Health and Human Development, IBN97201 from the
National Science Foundation, the Pediatric Epilepsy Research Center, and the Alz-
heimer’s Disease Research Center (National Institutes of Health/National Institute
on Aging Grant AG 05136), and the University of Washington (R.A.S.). We thank
Roger Baldwin, Don Shin, Tom Teal, and Dawit Neraio for their expert technical
assistance and Dr. Ulo Langel from Stockholm University for a kind gift of M35.
Correspondence should be addressed to Andrey M. Mazarati, Department of
Neurology, University of California Los Angeles School of Medicine, Veterans Affairs
Greater Los Angeles Healthcare System, 111N1 Epilepsy Research, 16111 Plummer
Street, Sepulveda, CA 91343-2099. E-mail: mailto:mazarati@ucla.edu.
Copyright © 2000 Society for Neuroscience 0270-6474/00/206276-06$15.00/0
The Journal of Neuroscience, August 15, 2000, 20(16):6276–6281
using the E14 cell line, and the colony has remained in-bred on the
129OlaHsd strain. The GalOE construct was made by linking a 5.8 kb
section of the human dopamine

-hydroxylase promoter (hDBH) (Mercer
et al., 1991) to a 4.6 kb section of the mouse galanin gene, containing the
entire galanin coding sequence. This construct was ligated into the plasmid
pBS271B.3C. The 10.4 kb targeting vector was liberated and injected into
the pronucleus of fertilized mouse eggs (SJL ⫻ C57BL6) with the services
of DNX Transgenic (Princeton, NJ). Of a total of 50 mice screened, four
founders carried the transgene. Three lines of mice were established, and
all mice used in these studies were from line 1923. This line was back-
crossed into the C57BL6/J strain for at least six generations, to remove
potential strain-dependent allelic variations that might contribute to be-
havioral and physiological differences. All GalOE mice used in these
studies and their WT controls were genotyped by dot hybridization of
genomic DNA to confirm the presence of the hDBH transgene, as previ-
ously described (Mercer et al., 1991). Overexpression of the galanin
transgene in specific brain regions of the 1923 line was confirmed by in situ
hybridization assays, as well as by radioimmunoassay for total brain galanin
content (Hohmann et al., 1997).
The experiments were performed with 12- to 20-week-old animals. Our
experiments always compared transgenic mice with WT controls of similar
genetic backgrounds. All experiments were approved by the Animal Re-
search Committee of the Veterans Affairs Greater Los Angeles Health
Care System.
Surgery. Mice were anesthetized with ketamine (60 mg/kg)/xylazine
(15 mg/kg) and stereotaxically implanted with a bipolar stimulating elec-
trode into the angular bundle of the perforant path (0.5 mm anterior and
2.5 mm lateral to lambda, 1.5–2.0 mm ventral from the brain surface) and
a bipolar recording electrode into the ipsilateral granule cell layer of the
dorsal dentate gyrus (2.0 mm posterior and 1.0 mm lateral to bregma,
1.5–2.0 mm ventral from brain surface). The final position of both elec-
trodes was optimized by finding a population spike with the amplitude of
at least 2 mV, evoked from the dentate gyrus by a stimulus delivered to the
perforant path (0.1 msec, 10 V square wave monophasic stimuli delivered
every 10 sec). This surgery protocol was used in the experiments with
SSSE, afterdischarge (AD) properties, long-term potentiation (LTP), and
paired-pulse inhibition. All in vivo experiments were performed in free-
running animals aftera7drecovery.
Seizure models. SSSE was induced by 5 sec trains (0.1 msec, 10 V, 33 Hz
square wave monophasic stimuli) delivered to the perforant path every
minute, together with continuous 3 Hz stimulation using the same param-
eters. Total seizure time (time spent in EEG seizures after the end of the
stimulation) and SE duration (time elapsed between end of stimulation
and termination of the last EEG seizure) were measured by means of
Harmony Software (Stellate Systems, Montreal, Quebec, Canada). Kainic
acid (KA) was administered subcutaneously in doses of 20 or 30 mg/kg.
The animals were killed3dafter KA, and the brains were sectioned and
stained with hematoxylin and eosin. Neuronal damage, evident as the
appearance of eosinophilic neurons with pyknotic nuclei (Sankar et al.,
1997), was assessed by an unbiased investigator in six 20-
m-thick sections
of the hippocampus from each animal. The extent of the damage was
described as minimal (occasional eosinophilic shrunken neurons were
found), mild (1 of 10 neurons was injured), moderate (up to half of the
neurons were injured), or severe (more than half of the neurons appeared
to be damaged). Pentylenetetrazol (PTZ) was administered intraperito-
neally in doses of 20–60 mg/kg. After injection of KA or PTZ, seizures
were videotaped for 2 hr and quantified off-line by an unbiased investiga-
tor, according to a modified Racine (1972a) scoring system: 0, no motor
seizures; 1, freezing, mouth, or facial movements; 2, head nodding or
isolated twitches; 3, unilateral/bilateral forelimb clonus; 4, rearing; 5,
rearing and falling; and 6, tonic seizure with hindlimb extension or death.
Intracerebral injections. For intracerebroventricular injections, the ani-
mals were chronically implanted bilaterally with guide cannulas into the
lateral ventricles. Rat galanin-[1–29] (American Peptide Company, Sunny-
vale, CA), a nonselective agonist for the galanin receptor subtypes 1, 2, and
3 (Bartfai et al., 1992), and M35, a mixed galanin receptor agonist/
antagonist, which possesses predominantly antagonistic effects in the dose
used (Antoniou et al., 1997), were injected using a Hamilton microsyringe
(0.5 nmol in 1
l over 5 min).
Studies of afterdischarge properties. To test AD threshold and duration,
2 sec trains of 1 msec square wave biphasic stimuli at 60 Hz were applied
to the perforant path, and the EEG response was recorded from the
dentate gyrus. Initial current intensity was 50
A with 50
A increments,
3 hr apart (Mazarati and Wasterlain, 1997a).
Glutamate accumulation. To measure glutamate accumulation, hip-
pocampi from mice that were killed were dissected on ice, cut coronally at
475
m, and allowed to recover for 3 hr in 20 ml chambers in ACSF.
Samples of 200
l were taken every minute for the first 5 min and then at
10, 20, and 30 min. After collecting baseline samples, slices were placed
into ACSF containing 60 m
M KCl to induce depolarization. Samples were
then collected at the same time intervals. The protein content was detected
by Lowry assay. Glutamate and aspartate were separated as O-phtalaldehyde
derivatives by HPLC (Baxter et al., 1991). In a separate set of experiments,
hippocampal slices from WT controls for GalKO (n ⫽ 3) were treated with
M35 (0.5
M) 10 min before 60 mM KCl-induced depolarization. Control
slices were treated with 0.9% saline instead of M35.
Long-term potentiation in the dentate g yrus in vivo. For test stimulations,
the population spike was evoked from the dentate gyrus by stimuli applied
to the perforant path (0.4 msec square wave monophasic stimuli delivered
every 10 sec starting at2Vwith0.2Vincrements); the intensity of stimulus
needed to induce half the maximal population spike was used for tetanic
stimulation (three trains of eight pulses for 0.4 msec at 400 Hz with a 10 sec
intertrain interval; Namgung et al., 1995). The population spike (PS)
amplitude and EPSP slope were measured as previously described
(Shirasaka and Wasterlain, 1994) as an averaged response to 10 consecu-
tive test stimulations delivered at 0.1 Hz, before, and 6 hr, 2, and4dafter
tetanic stimulation. The PS amplitude (in millivolts) was calculated as
[(field potential at the beginning of population spike ⫹ field potential at
the end of population spike)/2 ⫺ (field potential at the peak of population
spike)]. The EPSP slope (in millivolts per millisecond) was measured
between two fixed points after the EPSP onset and before the PS onset.
Paired-pulse inhibition. Pairs of 10 V, 0.1 msec square wave monophasic
0.1 Hz stimuli were delivered at 40 msec of 400 msec interstimulus
intervals (ISI) to study short ISI-dependent and long ISI-dependent inhi-
bition, respectively. Paired pulse inhibition was expressed as the ratio of
the average of 10 consecutive second PS (P2) to the average of 10
consecutive first PS (P1) (Shirasaka and Wasterlain, 1994).
Note. In certain cases, the same animals were used for two experiments.
Specifically, after the studies of LTP, AD properties, paired-pulse inhibi-
tion, or PTZ-induced convulsions, the mice were allowed to recover for 2
weeks, randomized, and used for studies of SSSE, KA-induced seizures or
glutamate accumulation.
RESULTS
We examined the ability of the transgenic mice to develop SSSE,
using a protocol similar to that used to study the action of galanin
in rats (Mazarati et al., 1998). Intermittent stimulation of the
perforant path, the major excitatory input from the entorhinal
cortex to the hippocampus (Heinemann et al., 1992), generates
seizures that are initially stimulus-bound, but with prolonged stim-
ulation become self-sustaining (Mazarati et al., 1998b). Whereas 30
min of perforant path stimulation (PPS) was not sufficient to induce
SSSE in any of WT, all of GalKO developed SSSE with the last
seizure observed 345 ⫾ 45 min after PPS. In contrast, 60 min PPS
induced SSSE in WT controls for GalOE, but only brief seizure
activity was observed in GalOE (the last seizure after PPS oc-
curred at 318 ⫾ 57 and 25 ⫾ 5 min, respectively; Fig. 1a–d shows
time spent in seizures after PPS).
To determine whether this role of galanin was unique to SSSE,
we used a further model of limbic status epilepticus (SE), the
systemic administration of KA (Nadler, 1981). In WT controls for
GalKO, KA (20 mg/kg, s.c.) induced repetitive stage 1–3 seizures
Figure 1. SSSE induced by PPS. Lef t, EEG in the dentate gyrus 30 min
after the end of PPS. Right, Time in seizures after PPS (mean ⫾ SEM). PPS
for 30 min was insufficient to induce SSSE in WT (A), but induced SSSE in
GalKO ( B). PPS for 60 min induced SSSE in WT controls for GalOE (C),
but had no effect in GalOE (D). E, Afterdischarge threshold; F, afterdis-
charge duration. For each group, n ⫽ 4. Data are presented as mean ⫾
SEM. *p ⬍ 0.05 versus respective WT control (Student’s t test).
Mazarati et al. • Seizures in Galanin Transgenic Mice J. Neurosci., August 15, 2000, 20(16):6276–6281 6277
and a few stage 5 seizures. Neuronal injury in the hippocampus was
restricted to the CA1, whereas the CA3 and dentate hilus were
spared (Table 1). In GalKO treated with this same dose of KA, the
incidence of stage 5 seizures was 17-fold higher than in WT, and
neuronal injury extended to the CA3 and hilus (Table 1). In WT
controls for GalOE, KA (30 mg/kg, s.c.) induced severe seizures,
which resulted in the death of five of six animals within 30 min. In
the single surviving animal, moderate injury was present in the
CA1, CA3, and hilus (Table 1). In GalOE this dose of KA induced
few stage 5 seizures, which were lethal in only one of six animals,
and survivors showed only mild injury to the hippocampus (Table 1).
Thus, the ability of animals to establish SE inversely correlated
with levels of brain galanin expression in two models of SE in two
different lines of mice, suggesting an important role for galanin as
an endogenous anticonvulsant.
To extend this conclusion to seizure models beyond SE, we
examined the AD properties in the perforant path-dentate granule
cell synapse and the severity of seizures induced by PTZ. De-
creased threshold and prolonged duration of AD reflect higher
hippocampal excitability and are often seen in chronic epilepsy
(Racine, 1972b; Shirasaka and Wasterlain, 1994; Mazarati and
Wasterlain, 1997a). GalKO showed a significantly longer AD du-
ration, and GalOE had a significantly higher AD threshold com-
pared to their respective WT (Fig. 1E,F). Seizures induced by
PTZ (20–60 mg/kg) were more severe in GalKO (Fig. 2A) and less
severe in GalOE (Fig. 2B) compared to their WT controls.
To confirm that alterations in seizure responsiveness in GalKO
and GalOE were mediated by galanin receptors, we examined the
effects of galanin receptor ligands on seizure severity. In GalKO,
galanin [0.5 nmol/side, bilaterally into lateral brain ventricles (in-
tracerebroventricularly)] delayed the occurrence of the first seizure
and attenuated the severity of PTZ (40 mg/kg)-induced seizures,
bringing both indices to the levels observed in WT (Fig. 2C). In
GalOE, the administration of M35, a partial galanin receptor
agonist, which would be expected to have predominantly antagonist
effects at the dose used (0.5 nmol; Antoniou et al., 1997), decreased
the latency and increased the severity of PTZ (50 mg/kg)-induced
seizures (Fig. 2D). Galanin acts presynaptically to block glutamate
release from rat hippocampal slices, possibly by opening ATP-
sensitive potassium channels (Zini et al., 1993a,b). Because gluta-
mate is a major excitatory neurotransmitter involved in limbic
epilepsy (Sloviter and Dempster, 1985), we tested the hypothesis
that the observed differences in susceptibility to seizure induction
between GalKO and GalOE are attributable to a differential reg-
ulation of glutamate release from the hippocampus in the two
mutant strains. There was no difference in basal accumulation of
glutamate in the bathing medium among the groups of transgenic
and WT animals (Fig. 3A,B). After KCl (60 m
M)-induced depolar-
ization, hippocampal slices from GalKO released significantly
more glutamate than slices from WT (Fig. 3A), whereas slices from
GalOE showed no response to the same depolarization challenge
(Fig. 3B). In hippocampal slices from WT controls for GalKO,
galanin receptor antagonist M35 (0.5
M), did not affect basal
glutamate release, but under conditions of KCl-induced depolar-
ization the peptide induced 30% higher increase of glutamate
release than in sham-treated slices ( p ⬍ 0.05).
To further elucidate the mechanisms that contribute to altered
seizure responses in the transgenic animals, we examined LTP in
the perforant path–dentate gyrus pathway that involved the same
morphological substrate as the one used to induce SSSE. Under
basal conditions, no differences in both PS amplitude and EPSP
slope were observed between WT (Fig. 4A,B) and transgenic
animals [half-maximal PS (in millivolts)/EPSP slope (millivolts per
millisecond) were 1.6 ⫾ 0.3/0.38 ⫾ 0.02 in GalKO, and 1.6 ⫾
0.2/0.37 ⫾ 0.01 in GalOE]. In both lines of WT, tetanic stimulation
applied to the perforant path induced a 1.6-fold increase in PS
amplitude and a 1.7-fold increase in the EPSP slope (Fig. 4A,B),
which persisted at2dafter LTP induction. In GalKO, the increase
in both PS amplitude and the EPSP slope was significantly greater
than in WT (2.4- and 2.2-fold respectively), and the latter was still
present4dafter tetanic stimulation (Fig. 4C,D). In addition, test
stimulations applied after tetanus induced multiple PS, which were
never observed in WT (Fig. 4E). Multiple PS reflect granule cell
hyperexcitability and are often observed in chronic epilepsy (Slo-
viter, 1991, 1992; Shirasaka and Wasterlain, 1994). In GalOE, the
Table 1. Kainic acid-induced seizures in GalKO and GalOE
transgenic mice
Group
(dose of KA)
Number of st. 5
seizures for 2 hrs
a
Mortality
Neuronal
injury
GalKO 69 ⫾ 7.3* 0/6 CA1: ⫹⫹⫹
(20 mg/kg) (45–85)
b
CA3: ⫹⫹
Hilus: ⫹⫹
WT control for KO 3.6 ⫾ 0.7 0/6 CA1: ⫹⫹⫹
(20 mg/kg) (2–6)
b
CA3: ⫺
Hilus: ⫺
GalOE 1.4 ⫾ 1* 1/6* CA1: ⫹
(30 mg/kg) (0–5)
b
CA3: ⫹
Hilus: ⫹
WT control for GalOE 7.2 ⫾ 1.5 5/6 CA1: (⫹⫹⫹)
c
(30 mg/kg) (3–11)
b
CA3: (⫹⫹⫹)
c
Hilus: (⫹⫹⫹)
c
⫺, no injured neurons were found; ⫹, minimal injury; ⫹⫹, mild injury;
⫹⫹⫹, moderate injury.
*p ⬍ 0.05 vs respective WT control (Mann–Whitney U test).
a
Mean ⫾ SEM.
b
Numbers in parentheses show minimal and maximal values.
c
Numbers in parentheses indicate neuronal injury in the only surviving animal in WT
controls for GalOE group.
Figure 2. PTZ-induced seizures in transgenic animals. Dose–response to
PTZ in GalKO (A) and GalOE (B). C, Bilateral intracerebroventricular
injection of galanin (0.5 nmol/side) to GalKO attenuated seizures induced
by PTZ (40 mg/kg) (GalKO ⫹ GA L), whereas similar administration of
M35 (0.5 nmol/side) to WT controls increased seizures (WT ⫹ M35).
D, GalOE responded with more severe PTZ-induced seizures (50 mg/kg)
when injected bilaterally intracerebroventricularly with M35 (0.5 nmol/side;
GalOE ⫹ M35). For each group, n ⫽ 6. Data are presented as mean ⫾
SEM. *p ⬍ 0.05 versus respective WT (Mann–Whitney U test for seizure
score and Student’s t test for seizure latency).
6278 J. Neurosci., August 15, 2000, 20(16):6276–6281 Mazarati et al. • Seizures in Galanin Transgenic Mice
initial increase in PS amplitude and the EPSP slope (6 hr after
tetanic stimulation) were similar to those in WT; however both
parameters had returned to baseline by2dafter LTP induction
(Fig. 4C,D). Therefore, GalKO showed increased LTP initiation
and maintenance compared to WT, whereas GalOE displayed a
more rapid LTP decay.
Finally, neither short ISI-dependent inhibition (GABA-A) nor
long ISI-dependent inhibition differed among WT, KO, and OE
animals (Fig. 5), suggesting that dentate circuits of feedback inhi-
bition (Sloviter, 1991, 1992; Shirasaka and Wasterlain, 1994) were
functional.
DISCUSSION
Our results suggest that galanin is a major endogenous modulator
of excitability in the mouse hippocampus under a broad variety of
physiological and pathological circumstances.
Previous studies in rats showed that the perforant path–dentate
gyrus pathway might be critical for the evolvement of SSSE (Vice-
domini and Nadler, 1987; Mazarati et al., 1998b). In our experi-
ments we focused primarily on the properties of the perforant
path-dentate granule cell synapse to address the importance of
endogenous galanin in regulating hippocampal excitability under
normal and pathological conditions. Indeed, strong differences
were observed between GalKO and GalOE in their ability to
develop SSSE, which inversely correlated with the level of galanin
expression. Further studies of perforant path–dentate gyrus path-
way properties in transgenic animals, revealed different responses
under conditions of nonseizure stimulation as well. Increased AD
duration in GalKO and increased AD threshold in GalOE suggest
that mice lacking galanin have higher excitability of perforant
path–dentate gyrus projection, whereas mice overexpressing gala-
nin are more resistant to such stimulation. These data are in
accordance with our recent observations that GalKO showed ac-
celerated perforant path kindling compared to their WT controls
(A. Mazarati and C. Wasterlain, unpublished data). Furthermore,
our studies showing enhanced ability of GalKO to maintain LTP
and faster LTP decay in GalOE provide additional confirmation for
galanin modulation of hippocampal excitability. It should be men-
tioned that LTP is enhanced soon after SSSE in rats (Mazarati and
Wasterlain, 1997a; Wasterlain et al., 1998) and that the agents that
block NMDA-dependent LTP also block SSSE (Mazarati et al.,
1999). Therefore, galanin-modulated synaptic potentiation may
underlie the observed differences in the ability to develop SSSE in
Figure 3. Glutamate accumulation from hippocampal slices of transgenic
mice. Samples were taken 5 min after bath application. Basal glutamate
accumulation did not vary significantly among the groups. A, During KCl-
induced depolarization, hippocampal slices from GalKO (n ⫽ 4) showed a
significantly greater glutamate accumulation than WT (n ⫽ 4). B, No
significant increase of glutamate accumulation was observed in GalOE (for
both GalOE and WT; n ⫽ 5). Data are presented as mean ⫾ SEM. *p ⬍
0.05 versus basal release (paired t test); †p ⬍ 0.05 versus WT (Student’s t
test). M35 (0.5
M) did not affect basal glutamate accumulation from
hippocampal slices of WT controls for GalKO (n ⫽ 3) but resulted in 30%
larger increase of glutamate accumulation under conditions of 60 mM KCl,
compared to sham-treated controls ( p ⬍ 0.05) (data not shown).
Figure 4. Long-term potentiation in the dentate gyrus of transgenic mice
in vivo. Half-maximal population spike (PS) amplitude (A) and EPSP slope
(B) after tetanic stimulation of perforant path in WT controls for GalKO
(open bars) and WT controls for GalOE (black bars) (mean ⫾ SEM). No
significant differences were observed between the two groups. Half-
maximal PS amplitude (C) and EPSP slope (D) in GalKO (open squares)
and GalOE (dashed triangles) compared to WT controls (black diamonds).
Data from the two WT groups were pooled, because they were not signif-
icantly different from one another. For each group, n ⫽ 4. *p ⬍ 0.05 versus
0 (before tetanus, repeated measures ANOVA ⫾ Neuman–Keuls test),
†p ⬍ 0.05 versus WT (Student’s t test). E, Sample responses to test
stimulations. Note the appearance of multiple PSs in GalKO after tetanus.
Figure 5. Paired pulse inhibition in the dentate gyrus of transgenic mice
in vivo. Left, Sample paired pulse tracings taken from individual animals at
40 and 400 msec ISI. Arrows indicate first (P1) and second (P2) PS (for 400
msec ISI the first PS is omitted because it is the same at 40 msec ISI). Right,
The ratio of P2 to P1 in the groups show no differences among transgenic
and WT animals (black bars, 40 msec; open bars, 400 msec ISI). The data
from WT controls for GalKO and GalOE are pooled because there were no
statistically significant differences between the two groups ( p ⬎ 0.05;
one-way ANOVA). Data are presented as mean ⫾ SEM.
Mazarati et al. • Seizures in Galanin Transgenic Mice J. Neurosci., August 15, 2000, 20(16):6276–6281 6279
transgenic animals. Previous studies showing that exogenously ad-
ministered galanin inhibits LTP in the CA1 of hippocampal slices
from guinea pigs (Sakurai et al., 1996) support this conclusion.
Hence, our experiments suggest that endogenous galanin mod-
ulates the physiological properties of the perforant path–dentate
granule complex, which commands the gate of entry into the
hippocampal trisynaptic excitatory loop and regulates hippocampal
excitability under both normal and pathological conditions.
To determine whether the differences between galanin trans-
genic animals were applicable to other types of seizures, we studied
the ability of the animals to develop status epilepticus after injec-
tion of KA and under conditions of acute PTZ-induced convul-
sions. The behavioral pattern of KA-induced limbic seizures in
GalKO and GalOE was compatible with the one during SSSE.
Milder character of neuronal hippocampal injury in GalOE and
greater severity of injury in GalKO confirm the differences in the
impact of KA-induced seizures on the hippocampus.
SSSE is dependent on the potentiation of excitatory glutamater-
gic synapses (Mazarati and Wasterlain, 1997b; Rice and De-
Lorenzo, 1998; Wasterlain et al., 1998, 1999). To outline the
possible biochemical substrate underlying the differences in hip-
pocampal physiology between GalKO and GalOE, we examined
glutamate release from hippocampal slices of two types of animals.
We found enhanced release of glutamate after depolarization in
hippocampi from the GalKO and the reduced release in the slices
from GalOE compared to the appropriate WT, suggesting that
galanin presynaptically inhibits glutamate release. These observa-
tions are compatible with the data showing attenuation of gluta-
mate release from rat hippocampal slices, possibly through opening
of ATP-dependent potassium channels (Zini et al., 1993a,b).
In contrast to SSSE, or KA-induced seizures, PTZ seizures
originate from the brainstem and medial thalamic nuclei (Miller
and Ferrendelli, 1988), where indeed galanin neurons are abundant
(Merchenthaler et al., 1993). PTZ-induced convulsions may repre-
sent either of two models of epilepsy depending on the dose used:
low doses of PTZ (20–40 mg/kg) induce clonic convulsions, a
model of petit mal seizures, whereas higher doses of PTZ induce
generalized tonic–clonic convulsions, a model of major motor
seizures (Woodburry, 1972). The leftward shift of the PTZ-seizure
dose–response curve in GalKO and its rightward shift in GalOE
compared to respective WT found in our studies extends the
anticonvulsant action of endogenous galanin to those two forms of
epilepsy. The results of the experiments involving PTZ also suggest
that the source of hippocampal modulation by galanin may be
extrahippocampal. Indeed, the abundant galanin-immunoreactive
fibers seen throughout the hippocampus are the axons of neurons
located in medial septum, locus coeruleus, and hypothalamus (Me-
lander et al., 1986), and expression of pre-progalanin mRNA is low
in all cell groups of the hippocampus (Melander et al., 1986; Cortes
et al., 1990; Merchenthaler et al., 1993).
The experiments with intracerebral injections of galanin recep-
tor ligands suggest that the observed differences between GalKO
and GalOE are mediated by galanin receptors. Intracerebroven-
tricular injection of galanin in the GalKO and of M35 in the GalOE
brought their seizure responses back toward the WT range. Simi-
larly, in hippocampal slices from WT animals, blocking of galanin
receptors by M35 increased KCl-induced glutamate release, mim-
icking the differences observed between WT and GalKO animals.
M35-induced increase of glutamate release was not as high as that
difference between WT and GalKO, which may be attributable to
either an incomplete blockade of galanin receptors or to a prefer-
ential blockade of one of the subtypes of galanin receptors (e.g.,
GalR1), leaving other subtype or subtypes (GalR2, GalR3) avail-
able for galanin action. On the other hand, in GalKO animals, none
of galanin receptor subtypes are functional because of the absence
of the endogenous ligand.
Interestingly, neither basal release of glutamate from hippocam-
pal slices, nor GABA
A
-mediated recurrent inhibition, as reflected
by paired pulse inhibition in the dentate gyrus (Shirasaka and
Wasterlain, 1994), were altered in galanin transgenic animals. Fur-
thermore, although GalKO mice had a lower seizure threshold,
they never developed spontaneous seizures. This finding suggests
that galanin has little influence on the hippocampus under resting
conditions and shows its modulatory effect only as a response to
repetitive or seizure-like stimulation. This conclusion is supported
by present and previous (Mazarati et al., 1998a) observations, that
M35 alone did not induce seizures in vivo, although it increased the
severity of electrically or chemically induced convulsions, and that
in vitro M35 did not alter basal glutamate release, but instead
increased glutamate release induced by depolarization.
The majority of previous studies of galanin have been performed
in the rat. However, the effects of galanin receptor ligands on
seizures observed in our experiments along with the pattern of
altered responses in galanin transgenic animals, suggest that basic
properties of galanin in mice are comparable with those in rats (as
well as guinea pigs; Sakurai et al., 1996). Thus, galanin transgenic
mice provide a useful tool for studies of galanin physiology.
Several neuropeptides have been implicated in modulation of
hippocampal function and seizure activity (Marksteiner et al.,
1990; Drake et al., 1994; Harrison et al., 1995; Liu et al., 1999b;
Mazarati et al., 1999). The advances in molecular biology that have
allowed construction of transgenic animals have introduced a novel
approach to analyze the role of these peptides in seizures. Recent
findings using transgenic mice with NPY (Baraban et al., 1997) or
substance P (Liu et al., 1999a) mutations revealed a critical role of
these two peptides in seizures. The present report shows an im-
portant role of galanin as a putative “endogenous anticonvulsant.”
Taken together, these findings suggest that although there is no
“ultimate” proepileptic or antiepileptic peptide, very fine abnor-
malities in peptide-modulated tuning of hippocampal functioning
as a result of inherited or acquired defects may strongly affect
predisposition of the brain to epilepsy. As a result, neuropeptide
receptors may become a target for the development of new anti-
epileptic drugs.
In conclusion, galanin may counteract excess excitation in re-
sponse to physiological or pathological stimuli and may offer a
mechanism by which deep brain nuclei modulate hippocampal
function and excitability. An intriguing speculation is that inher-
ited defects in the expression of this endogenous anticonvulsant
may be epileptogenic in animals or humans. As a result, galanin
agonists have the potential of making excellent anticonvulsants,
because they may be able to inhibit a broad variety of seizures in
the pathologically activated hippocampus while relatively sparing
normal brain function.
REFERENCES
Antoniou K, Kehr J, Snitt K, Ogren SO (1997) Differential effects of the
neuropeptide galanin on striatal acetylcholine release in anaesthetized
and awake rats. Br J Pharmacol 121:1180–1186.
Baraban SC, Hollopeter G, Erickson JC, Schwartzkroin PA, Palmiter RD
(1997) Knock-out mice reveal a critical antiepileptic role for neuropep-
tide Y. J Neurosci 17:8927–8936.
Bartfai T, Fisone G, Langel U (1992) Galanin and galanin antagonists:
molecular and biochemical perspectives. Trends Pharmacol Sci
13:312–317.
Bartfai T, Ho¨kfelt T, Langel U
¨
(1993) Galanin: a neuro-endocrine pep-
tide. Crit Rev Neurobiol 7:229–274.
Bauer FE, Ginsberg L, Venetikou M, MacKay DJ, Burrin JM, Bloom SR
(1986) Growth hormone release in man induced by galanin, a new
hypothalamic peptide. Lancet 2:192–195.
Baxter CF, Oh CC, Wasterlain CG, Ozaki LK, Baldwin RA (1991) Al-
terations of GABA metabolism and seizure susceptibility in the substan-
tia nigra of the kindled rat acclimating to changes in osmotic state.
Neurochem Res 16:269–278.
Cortes R, Ceccatelli S, Schalling M, Hokfelt T (1990) Differential effects
of intracerebroventricular colchicine administration on the expression of
mRNAs for neuropeptides and neurotransmitter enzymes, with special
emphasis on galanin: an in situ hybridization study. Synapse 6:369–391.
Drake CT, Terman GW, Simmons ML, Milner TA, Kunkel DD, Schwartz-
kroin PA, Chavkin C (1994) Dynorphin opioids present in dentate
granule cells may function as retrograde inhibitory neurotransmitters.
J Neurosci 14:3736–3750.
Harrison MB, Shumate MD, Lothman EW (1995) Opioid peptide expres-
sion in models of chronic temporal lobe epilepsy. Neuroscience
65:785–795.
Heinemann U, Beck H, Dreier JP, Ficker E, Stabel J, Zhang CL (1992)
6280 J. Neurosci., August 15, 2000, 20(16):6276–6281 Mazarati et al. • Seizures in Galanin Transgenic Mice
The dentate gyrus as a regulated gate for the propagation of epileptiform
activity. In: The dentate gyrus and its role in seizures (Ribak CE, Gall
CM, Mody I, eds) pp 273–280. Amsterdam: Elsevier.
Hohmann JG, Cadd GC, Teal TH, Clifton DK, Steiner RA (1997) Trans-
genic mice that overexpress the galanin gene in brainstem neurons. Soc
Neurosci Abstr 23:1878.
Kerr BJ, Cafferty WB, Gupta YK, Bacon A, Wynick D, McMahon SB,
Thompson SW (2000) Galanin knockout mice reveal nociceptive defi-
cits following peripheral nerve injury. Eur J Neurosci 12:793–802.
Lamour Y, Senut MC, Dutar P, Bassant MH (1988) Neuropeptides and
septo-hippocampal neurons: electrophysiological effects and distributions
of immunoreactivity. Peptides 9:1351–1359.
Liu H, Cao Y, Basbaum AI, Mazarati AM, Sankar R, Wasterlain CG
(1999a) Resistance to excitotoxin-induced seizures and neuronal death
in mice lacking the preprotachykinin A gene. Proc Natl Acad Sci USA
96:12096–12101.
Liu H, Mazarati AM, Katsumori H, Sankar R, Wasterlain CG (1999b)
Substance P is expressed in hippocampal principal neurons during status
epilepticus and plays a critical role in the maintenance of status epilep-
ticus. Proc Natl Acad Sci USA 96:5286–5291.
Marksteiner J, Ortler M, Bellmann R, Sperk G (1990) Neuropeptide Y
biosynthesis is markedly induced in mossy fibers during temporal lobe
epilepsy of the rat. Neurosci Lett 112:143–148.
Mazarati A, Wasterlain C (1997a) Loss of hippocampal inhibition and
enhanced LTP after self-sustaining status epilepticus. Epilepsia [Suppl]
38:178.
Mazarati AM, Wasterlain CG (1997b) Selective facilitation of kindled
seizures from the amygdala after hippocampal lesions induced by per-
forant path stimulation. Neurosci Lett 224:165–168.
Mazarati AM, Wasterlain CG (1999) N-methyl-D-aspartate receptor an-
tagonists abolish the maintenance phase of self-sustaining status epilep-
ticus in rat. Neurosci Lett 265:187–90.
Mazarati AM, Halaszi E, Telegdy G (1992) Anticonvulsive effects of ga-
lanin administered into the central nervous system upon the picrotoxin-
kindled seizure syndrome in rats. Brain Res 589:164–166.
Mazarati A, Liu H, Soomets U, Sankar R, Shin D, Katsumori H, Langel U,
Wasterlain CG (1998a) Galanin modulation of seizures and seizure
modulation of hippocampal galanin in animal models of status epilepti-
cus. J Neurosci 18:10070–10077.
Mazarati AM, Wasterlain CG, Sankar R, Shin D (1998b) Self-sustaining
status epilepticus after brief electrical stimulation of the perforant path.
Brain Res 801:251–253.
Mazarati AM, Liu H, Wasterlain CG (1999) Opioid peptide pharmacol-
ogy and immunochemistry in an animal model of self-sustaining status
epilepticus. Neuroscience 89:167–173.
Melander T, Staines WA, Rokaeus A (1986) Galanin-like immunoreactiv-
ity in hippocampal afferents in the rat, with special reference to cholin-
ergic and noradrenergic inputs. Neuroscience 19:223–240.
Mercer EH, Hoyle RP, Kapur RP, Brinster RL, Palmiter RD (1991) The
dopamine

-hydroxylase gene promoter directs expression on E.Coli lacZ
to sympathetic and other neurons in adult transgenic mice. Neuron
7:703–716.
Merchenthaler T, Lopez FJ, Negro-Vilar A (1993) Anatomy and Physiol-
ogy of central galanin-containing pathways. Prog Neurobiol 40:711–769.
Miller JW, Ferrendelli JA (1988) Brain stem and diencephalic structures
regulating experimental generalized (pentylenetetrazol) seizures in ro-
dents. In: Anatomy of epileptogenesis (Meldrum BS, Ferrendelli JA,
Wieser HG, eds) pp 57–69. London: John Libbey.
Nadler JV (1981) Minireview. Kainic acid as a tool for the study of
temporal lobe epilepsy. Life Sci 29:2031–2042.
Namgung U, Valcourt E, Routtenberg A (1995) Long-term potentiation
in vivo in the intact mouse hippocampus. Brain Res 689:85–92.
Racine RJ (1972a) Modification of seizure activity by electrical stimula-
tion: motor seizures. Electroencephalogr Clin Neurophysiol 32:281–294.
Racine RJ (1972b) Modification of seizure activity by electrical stimula-
tion. I. Afterdischarge threshold. Electroencephalogr Clin Neurophysiol
32:269–279.
Rice AC, DeLorenzo RJ (1998) NMDA receptor activation during status
epilepticus is required for the development of epilepsy. Brain Res
782:240–247.
Sakurai E, Maeda T, Kaneko S, Akaike A, Satoh M (1996) Galanin
inhibits long-term potentiation at Schaffer collateral-CA1 synapses in
guinea-pig hippocampal slices. Neurosci Lett 212:21–24.
Sankar R, Shin D, Wasterlain CG (1997) Serum neuron- specific enolase is
a marker for neuronal damage following status epilepticus in the rat.
Epilepsy Res 28:129–136.
Senut MC, Menetrey D, Lamour Y (1989) Cholinergic and peptidergic
projections from the medial septum and the nucleus of the diagonal band
of Broca to dorsal hippocampus, cingulate cortex and olfactory bulb: a
combined wheatgerm agglutinin-apohorseradish peroxidase-gold immu-
nohistochemical study. Neuroscience 30:385–403.
Shirasaka Y, Wasterlain CG (1994) Chronic epileptogenicity following
focal status epilepticus. Brain Res 655:33–44.
Skofitsch G, Jacobowitz DM (1985) Immunohistochemical mapping of
galanin-like neurons in the rat central nervous system. Peptides
6:509–546.
Skofitsch G, Jacobowitz DM (1986) Quantitative distribution of galanin-
like immunoreactivity in the rat central nervous system. Peptides
7:609–613.
Sloviter RS (1991) Permanently altered hippocampal structure, excitability
and inhibition after experimental status epilepticus in the rat: the “dor-
mant basket cell” hypothesis and its possible relevance to temporal lobe
epilepsy. Hippocampus 1:41–46.
Sloviter RS (1992) Possible functional consequences of synaptic reorgani-
zation in the dentate gyrus of kainate-treated rats. Neurosci Lett
137:91–96.
Sloviter RS, Dempster DW (1985) Epileptic brain damage is replicated
qualitatively in the rat hippocampus by central injection of glutamate and
aspartate but not by GABA and acetylcholine. Brain Res Bull 15:39–60.
Vicedomini JP, Nadler JV (1987) A model of status epilepticus based on
electrical stimulation of hippocampal afferent pathways. Exp Neurol
96:681–691.
Wasterlain CG, Mazarati AM, Sankar R, Shirasaka Y, Thompson K (1998)
Potentiation of kindling and long-term potentiation by hippocampal
lesions. In: Kindling 5. (Corcoran M, Moshe M, eds) pp 501–510. New
York: Plenum.
Woodburry DM (1972) Applications to drug evaluations. In: Experimen-
tal models of epilepsy (Purpura DP, Penry JK, Tower DB, Walter RB,
eds) pp 557–585. New York: Raven.
Wynick D, Small CJ, Bacon A, Holmes FE, Norman M, Ormandy CJ, Kilic
E, Kerr NC, Ghatei M, Talamantes F, Bloom SR, Pachnis V (1998)
Galanin regulates prolactin release and lactrotroph proliferation. Proc
Natl Acad Sci USA 95:12671–12676.
Zini S, Roisin M, Langel U, Bartfai T, Ben-Ari Y (1993a) Galanin re-
duces release of endogenous excitatory amino acids in the rat hippocam-
pus. Eur J Pharmacol 245:1–7.
Zini S, Roisin MP, Armengaud C, Ben-Ari Y (1993b) Effects of potas-
sium channels moduators on the release of glutamate induced by
ischaemic-like conditions in rat hippocampal slices. Neurosci Lett 153:
202–205.
Mazarati et al. • Seizures in Galanin Transgenic Mice J. Neurosci., August 15, 2000, 20(16):6276–6281 6281
