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The Journal of Neuroscience, August 15, 2000, 20(16):6276-6281
Modulation of Hippocampal Excitability and Seizures by
Galanin
Andrey M.
Mazarati1, 4,
John G.
Hohmann6, 8,
Andrea
Bacon5,
Hantao
Liu1, 4,
Raman
Sankar2, 3,
Robert A.
Steiner6, 7, 8,
David
Wynick5, and
Claude G.
Wasterlain1, 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
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ABSTRACT |
Previous studies have shown that the expression of the neuropeptide
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 enhanced
perforant path-dentate gyrus long-term potentiation (LTP) in GalKO and
reduced LTP in GalOE. GalKO showed increased duration of afterdischarge
(AD) evoked from the dentate gyrus by perforant path simulation,
whereas GalOE had increased threshold for AD induction.
Depolarization-induced glutamate 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 genetically 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
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INTRODUCTION |
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 neuroendocrine regulator of hypothalamo-adrenal
hormone release, feeding 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; Merchenthaler 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 epilepticus (SSSE) (Mazarati et al.,
1998b); the injection of galanin into the hippocampus attenuates
seizure activity, whereas galanin antagonists facilitate it (Mazarati
et al., 1992, 1998a). However, the functional significance of
endogenous galanin in regulating hippocampal 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 galanin 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 measured 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 physiological and pathological roles
of endogenous galanin.
We provide evidence that altered expression of the galanin gene affects
both hippocampal excitability and predisposition to seizures,
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.
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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 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
backcrossed into the C57BL6/J strain for at least six generations, to
remove potential strain-dependent allelic variations that might
contribute to behavioral 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 previously 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
Research 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 electrode 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 electrodes 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 after a 7 d recovery.
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 parameters. 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 killed 3 d after 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-µm-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 intraperitoneally 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
investigator, 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 animals were chronically implanted bilaterally with guide cannulas into the lateral ventricles. Rat galanin-[1-29] (American Peptide Company, Sunnyvale, 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 µl
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 µA with 50 µA increments, 3 hr apart (Mazarati and
Wasterlain, 1997a).
Glutamate accumulation. To measure glutamate accumulation,
hippocampi from mice that were killed were dissected on ice, cut coronally at 475 µm, and allowed to recover for 3 hr in 20 ml chambers in ACSF. Samples of 200 µl 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 mM 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 µM) 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 gyrus 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 at
2 V with 0.2 V increments); 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 consecutive test stimulations delivered at 0.1 Hz, before, and 6 hr, 2, and 4 d after 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 inhibition, 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 inhibition, 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.
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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 stimulation 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 occurred at 318 ± 57 and 25 ± 5 min,
respectively; Fig. 1a-d shows
time spent in seizures after PPS).
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Figure 1.
SSSE induced by PPS. Left, 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, afterdischarge duration.
For each group, n = 4. Data are presented as mean
± SEM. *p < 0.05 versus respective
WT control (Student's t test).
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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 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. Decreased 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 duration, and GalOE had a significantly higher
AD threshold compared 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.
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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 + GAL), 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).
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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 (intracerebroventricularly)] 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 glutamate 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 regulation 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 mM)-induced depolarization, 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 µM), did not affect basal
glutamate release, but under conditions of KCl-induced depolarization
the peptide induced 30% higher increase of glutamate release than in
sham-treated slices (p < 0.05).
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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 µM) 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).
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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 at 2 d after 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 present 4 d after 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 (Sloviter, 1991, 1992; Shirasaka and Wasterlain, 1994). In GalOE, the 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 by 2 d after LTP induction (Fig. 4C,D). Therefore, GalKO showed increased LTP
initiation and maintenance compared to WT, whereas GalOE displayed a
more rapid LTP decay.
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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 significantly 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.
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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 inhibition (Sloviter, 1991, 1992; Shirasaka and Wasterlain,
1994) were functional.
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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.
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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 (Vicedomini and
Nadler, 1987; Mazarati et al., 1998b). In our experiments 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 pathway 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 galanin are
more resistant to such stimulation. These data are in accordance with
our recent observations that GalKO showed accelerated 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 mentioned 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 transgenic animals. Previous studies showing that
exogenously administered 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 modulates 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 transgenic animals
were applicable to other types of seizures, we studied the ability of
the animals to develop status epilepticus after injection of KA and
under conditions of acute PTZ-induced convulsions. 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 glutamatergic
synapses (Mazarati and Wasterlain, 1997b; Rice and DeLorenzo, 1998;
Wasterlain et al., 1998, 1999). To outline the possible biochemical
substrate underlying the differences in hippocampal 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 observations
are compatible with the data showing attenuation of glutamate 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 represent 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 (Melander 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 receptor
ligands suggest that the observed differences between GalKO and GalOE
are mediated by galanin receptors. Intracerebroventricular injection of
galanin in the GalKO and of M35 in the GalOE brought their seizure
responses back toward the WT range. Similarly, in hippocampal slices
from WT animals, blocking of galanin receptors by M35 increased
KCl-induced glutamate release, mimicking 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 preferential blockade of one of the subtypes of galanin receptors
(e.g., GalR1), leaving other subtype or subtypes (GalR2, GalR3)
available 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 hippocampal
slices, nor GABAA-mediated recurrent inhibition,
as reflected by paired pulse inhibition in the dentate gyrus (Shirasaka
and Wasterlain, 1994), were altered in galanin transgenic animals. Furthermore, 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 important 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 abnormalities 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 antiepileptic drugs.
In conclusion, galanin may counteract excess excitation in response 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 inherited 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.
| |
FOOTNOTES |
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
Alzheimer'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{at}ucla.edu.
| |
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ABSTRACT
INTRODUCTION
Gene
