Epilepsia, 47(2):240–246, 2006
Blackwell Publishing, Inc.
C 2006 International League Against Epilepsy
Serotonin Depletion Attenuates AY-9944–Mediated Atypical
∗ †Eduard Bercovici, †‡Miguel A. Cortez, †Xiaomei Wang, and ∗ †‡O. Carter Snead III
∗ Institute of Medical Science, University of Toronto, †Brain and Behaviour Program, Division of Neurology, The Hospital for Sick
Children, and ‡Department of Pediatrics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
Summary: Purpose: To test the hypothesis that serotonin (5- duration in AY-treated rats compared with controls (p < 0.01).
HT) plays a role in the modulation of experimental atypical ab- HPLC data confirmed the PCPA depletion of 5-HT and 5-HIAA
sence seizures. in cortex, thalamus, hippocampus, and brainstem compared with
Methods: Male Long-Evans hooded rats were treated from ı
na¨ve rats. AY-treated rats showed higher levels of 5-HIAA and
postnatal day (P) 2 to P20 with the cholesterol inhibitor AY-9944 5-HT in the same brain regions, with a concomitant decrease in
(AY). Epidural electrodes were implanted for electrocorticog- rates of serotonin turnover.
raphy (ECoG) followed by serotonin depletion by using para- Conclusions: The data indicate that serotonin depletion pro-
cholorophenylalanine (PCPA). High-performance liquid chro- tects against experimental atypical absence seizures. The in-
matography (HPLC) was used to measure the levels of serotonin creased levels of 5-HIAA and 5-HT and altered rates of sero-
and its metabolite (5-HIAA) in various brain regions. Serotonin tonin turnover suggest that the serotonergic neurotransmission
metabolism was computed by using the 5-HIAA/5-HT ratio and may be perturbed in the AY rat. Key Words: Atypical absence
used to ascertain differences between groups. seizures—AY-9944—ECoG—5-HT—PCPA.
Results: PCPA treatment was associated with a significant
decrease in the total slow spike-and-wave discharge (SSWD)
Atypical absence seizures (AASs) are clinically dis- rent, bilaterally synchronous generalized 5- to 7-Hz SS-
tinct from typical absence (petit mal) seizures in terms of WDs that emanate from the cortex, the thalamus, and the
cognitive outcome, ictal behavior, and electroencephalo- hippocampus (7). AY-treated animals move about inter-
graphic (EEG) manifestations (1–3). Unlike typical ab- mittently during the seizure, and, unlike that in animal
sence seizures, AASs often occur as a component of se- models of typical absence seizures (8), the ictal behavior
vere, medically refractory childhood epilepsy syndromes, is not time-locked with the epileptiform discharge. Rather,
such as the Lennox–Gastaut syndrome or other malig- a poor correlation exists between the timing of the onset
nant epilepsy syndromes that are characterized by multiple and offset of the SSWD and the onset and offset of the
seizure types (4). AASs rarely occur alone; they almost al- behavioral ictal changes. In addition, unlike animal mod-
ways coexist with tonic seizures during sleep, myoclonic els of typical absence seizures, the AY model of AASs is
jerks, and generalized tonic–clonic seizures. The recur- characterized by cognitive impairment (9, 10).
rent, bilaterally synchronous 1.5- to 2.5-Hz slow spike- The administration of AY during rat brain development
and-wave discharge (SSWD) that typifies AASs is highly leads to a prepubertal onset of SSWDs (11) that persists
predictive of a poor cognitive outcome (5), and AASs are during the adult period, although the brain cholesterol re-
almost always associated with cognitive deficits (6). turns to normal values (12). The SSWDs in the AY model
The clinical ictal events observed in patients with are state dependent, going from intermittent bilaterally
Lennox–Gastaut syndrome in both awake and sleep states synchronous discharges during the awake state to a con-
are highly reproducible in the AY-9944 (AY) cholesterol tinuous SWD during slow-wave sleep, which is often in-
inhibition model. The electrocorticographic (ECoG) sig- terrupted by myoclonic jerks (13).
nature of the AY model consists of spontaneously recur- Administration of the precursor of serotonin, 5-
hydroxytryptophan (5-HTP), is associated with the in-
Accepted September 5, 2005. duction of myoclonic jerks in guinea pigs (14). These
Address correspondence and reprint requests to Dr. O.C. Snead myoclonic jerking movements can be prevented by pre-
III at Division of Neurology, The Hospital for Sick Children, 555
University Avenue, Toronto, Ontario, M5G 1X8, Canada. E-mail: treatment with para-chlorophenylalanine (PCPA) (15), a
firstname.lastname@example.org compound that depletes brain serotonin by selectively and
SEROTONIN DEPLETION AND ATYPICAL ABSENCE SEIZURES 241
irreversibly inhibiting tryptophan hydroxylase, the rate- Drugs
determining step in serotonin synthesis (16). Recently AY-9944 [trans-1,4-bis(2-chloro-benzylaminomethyl)
it was shown that selective serotonin reuptake inhibitors cyclohexane dihydrochloride] was a gift from Wyeth-
(SSRIs) exacerbate typical absence seizures, presumably Ayerst (Philadelphia, PA, U.S.A.). Para-chloropheny-
by the increase in serotonergic activity (17). lalanine methyl ester (PCPA) was purchased from Sigma
To date, inconsistent results have been seen regard- (Oakville, Ontario, Canada). Drugs and chemicals used for
ing the effect of serotonin on absence seizures. For ex- biochemical assay were purchased from Sigma and were
ample, in the genetic absence epilepsy rat from Stras- of the highest purity (HPLC grade). AY-9944 and PCPA
bourg (GAERS), decreasing serotonin via PCPA had no were both dissolved in distilled water. All drugs were in-
effect, nor did using 5-HTP or methysergide, a nonspecific jected in a volume of 2 ml/kg of body weight, unless stated
mixed agonist/antagonist (18). With the WAG/Rij absence otherwise.
seizure model, Coenen and van Luijtelaar (19) reported
that serotonin may indirectly modulate absence seizures
After birth, rat pups were randomly divided into two
via glutamate transmission. However, recent work with
groups, receiving subcutaneous AY (7.5 mg/kg in 1 ml/kg
the WAG/Rij model has shown that SSRIs may exacer-
of body weight) every 6 days from P2 to P20 (AY group,
bate absence seizures, and this may be mediated via the
n = 12) or the equivalent volume of distilled water (con-
5-HT2C receptor subtype. Thus little work demonstrates
trol group = 8) as previously described (7). No female
a direct relation between the effect of specific serotonin-
rats were used because of our previous finding that the
receptor subtypes and rodent absence seizures. To date,
female estrus cycle does modulate the SSWD duration in
the effect of serotonin on the AASs in the AY model has
the AY model (11). All rats were randomly divided into
not been elucidated. Because no receptor subtypes have
two groups: the first group included the AY rats treated
been implicated in the AY model, our goal was to de-
with either PCPA (n = 6) or vehicle (n = 6); and the sec-
termine the outcome of global depletion of serotonin via
ond group of control (Ctrl) rats (n = 8) that were given the
PCPA. Serotonin depletion via administration of PCPA
same PCPA treatment to ascertain any effect of serotonin
was chosen because this is widely used and very well
depletion on AY-SSWD and on the spontaneous burst ob-
characterized (16). Neurotoxic serotonin-depleting agents
served in controls (7).
(5,7-dyhydroxytryptamine, 5,7-DHT) are reliable but pro-
duce profound permanent axonal damage (20). Thus we Surgery and electrocorticography
chose to use PCPA because its effects are well described On surgery day at P60, all animals received intraperi-
and reversible (16). toneal (i.p.) atropine methyl bromide (0.5 mg/kg of body
Because of the occurrence of state-dependent changes weight) as a preanesthetic agent to control secretions.
in the EEG and the occurrence of myoclonic jerks during Fifteen minutes later, animals were anesthetized with a
sleep in AY-treated animals, we hypothesized that pertur- single i.p. injection of Somnotol (sodium pentobarbital,
bation of the serotonergic system may play a role in the 35 mg/kg), which provides anesthesia that lasts approx-
genesis of AASs in the AY model. Therefore the objective imately 2 to 3 hours. Surgeries were done in the animal
of this study was to determine the effect of PCPA-mediated facility with sterile equipment, and the greatest care was
serotonin depletion on SSWD duration in the AY model taken to ensure minimal pain and discomfort. Rats were
of AASs. implanted with two monopolar epidural electrodes placed
over the left and right frontal cortical regions and two
placed over the left and right parietal regions. Four watch-
maker screws were set in the lateral regions of the skull,
Animals and the preparation was secured with dental cement. Af-
Male Long-Evans hooded rats (250–300 g) and un- ter surgery, all animals were monitored daily by veterinary
timed pregnant Long-Evans hooded rats were obtained technicians for 5 days of recovery.
from Charles River (St. Constant, Quebec, Canada) and Electrocorticographic (ECoG) recordings were per-
housed in the animal facility at the Hospital for Sick Chil- formed in unrestrained animals in individual Plexiglas
dren (Toronto, Ontario, Canada). The suckling rat pups chambers lined with bedding (Harvard Apparatus, Hol-
were weaned at postnatal (P) day 21 and grouped in pairs. liston, MA, U.S.A.). A 20-min adaptation period was
Animals were kept in a controlled environment at a 12-h given before every ECoG to minimize movement arti-
light–dark cycle with lights on at 0600 h and ad lib access fact. Paper ECoG recordings were made by using a Grass
to food and water. All animal procedures were approved Polysomnograph (78D; Grass Instruments, Quincy, MA,
by the Animal Care Committee at the Hospital for Sick U.S.A.). Frontal and parietal differential recordings were
Children, which conforms to the rules and regulations of performed on two distinct channels. ECoG recordings
the Canadian Council on Animal Care and Animals for were made between 1000 and 1400 h to prevent circadian
Research Act (Ottawa, Canada). variations (21).
Epilepsia, Vol. 47, No. 2, 2006
242 E. BERCOVICI ET AL.
Experimental design within-subject variables to test the effect of PCPA in AY-
Each rat was monitored with a 1-h baseline ECoG treated rats as compared with vehicle (interaction effect).
recording 1 week after surgery day. On the following 3 Specific comparisons were made by using paired Student’s
consecutive days, PCPA (150 mg/kg in a volume of 2 t test to test statistical significance within each group. All
ml/kg) or vehicle was administered between 0900 and analyses were performed by using SigmaStat 2.0, and an
1100 h during the peak of serotonin levels (22). Then a test alpha of p = 0.05 was considered to be significant.
ECoG recording was obtained from each rat, 24 h after the
last PCPA injection.
Within the same chromatogram, sample peak areas
Brain serotonin analysis were compared with an internal standard to yield the
After the test ECoG recording, all rats were anesthetized corrected amount of neurotransmitter and presented as
deeply with pentobarbital (65 mg/kg). The brains were re- picograms/milligram of wet brain tissue. In addition to
moved and quickly dissected on ice. Thalamus, hippocam- measuring the levels of serotonin, we decided to study
pus, frontal–parietal cortex, and brainstem were individ- the functional activity of serotonin metabolism. Previous
ually stored in 1.5-ml microcentrifuge tubes and frozen studies showed that the ratio of serotonin metabolite (5-
at −80◦ C (23). Biochemical analysis of serotonin levels HIAA) to serotonin (5-HT) can serve as an index of the
was performed as described by Fletcher et al. (24). Tissue serotonin turnover rate (25). Thus we used the 5-HIAA/5-
was homogenized in 5 volumes of 0.2N perchloric acid HT ratio as an approximation of the serotonin turnover
(HClO4 ) containing 100 ng/ml of dihydroxybenzylamine (26,27).
(DHBA) as the internal standard. Homogenates were cen- Statistical analysis
trifuged at 20,800 g for 15 min at 4◦ C. The resulting su- Statistical analyses of biochemical results were per-
pernatant aliquots (20 μl) were filtered by using 0.22-μm formed only within the same brain region and for the
syringe filters and analyzed for serotonin and its metabo- same neurotransmitter/turnover rate. One-way ANOVAs
lite 5-hydroxyindoleacetic acid (5-HIAA) by using high- were used to analyze differences within each brain region.
performance liquid chromatography with electrochemical Comparisons were made a priori by using two-tailed un-
detection (HPLC-ED) (ESA, Bedford, MA, U.S.A.). The paired Student’s t tests. Specific comparisons were made
analytic system consisted of a 100-μl sample loop (Rheo- ı
between na¨ve rats and AY-vehicle–treated rats and also
dyne, Rhonert Park, CA, U.S.A.), a 3.2 × 150 mm, 3 between PCPA-treated rats and their respective controls.
μm C18 reverse phase column C18 reverse phase column For all statistical analyses, an alpha of p = 0.05 was used
(MD-150, ESA), and a pulse dampener. Electrochemical as a measure of significance.
detectors consisted of ESA Coulochem II detector with
5020 guard cell set at +350 mV and 5014B analytical RESULTS
cells set at potential −150 mV (electrode 1) and +300 mV
(electrode 2). Data were collected and integrated by us- AY-induced atypical absence seizures
ing the ESA 500 Chromatography Data System. The mo- AY treatment resulted in spontaneous, recurrent, bilat-
bile phase consisted of 75 mM sodium dihydrogen phos- erally synchronous 5- to 6-Hz SSWDs associated with
phate monohydrate, 1.7 mM octanesulfonic acid (sodium AASs, as previously described (7). AY-induced AASs
salt), 25 μM EDTA, and 8% acetonitrile adjusted to pH were characterized by staring, facial myoclonus, and
3.00 with phosphoric acid. Stock solutions of 5-HT and whisker twitching, with a gradual onset and disappearance
5-HIAA (HPLC grade, Sigma) were used to prepare ex- that usually began after the onset of the SSWDs and clearly
ternal standard curves. Data were collected and integrated outlasted them. The complete immobility or “frozen stare”
by using the ESA 500 Chromatography Data System. that typifies pharmacologic and genetic rat models of typ-
ical absence seizures was not observed in the AY-treated
Data analysis rats. Instead, the AY-treated animals showed an ability to
move intermittently during the seizures.
Measure of SSWDs
The total duration of AY-SSWDs and the spontaneous PCPA treatment–attenuated SSWDs in the AY model
bursting activity of controls were quantified over 1-h base- As shown in Fig. 1, PCPA treatment significantly re-
line and test ECoG recordings. AY-SSWDs were scored duced the total SSWD duration, as demonstrated by an
only if they appeared on both channels simultaneously interaction effect (p < 0.001, two-way repeated ANOVA).
at the frequency of 5–6 Hz with at least threefold higher Specifically, PCPA reduced total SSWDs from 484.42 ±
amplitude compared with baseline. Total AY-SSWD du- 63.98 to 259.42 ± 13.48 s/h (p < 0.01, Student’s t test).
ration was expressed as mean ± standard error of the No difference was observed when AY-treated rats were
mean (SEM). A two-way repeated analysis of variance given vehicle. Similarly, no significant difference was ob-
(ANOVA) was used with treatment (PCPA or vehicle) as served when control naive male rats were given a regimen
the between-subject and session (baseline or post) as the of PCPA.
Epilepsia, Vol. 47, No. 2, 2006
SEROTONIN DEPLETION AND ATYPICAL ABSENCE SEIZURES 243
noted in serotonin-turnover rates in both the thalamus and
When PCPA was administered to the GAERS model
of chronic typical absence epilepsy, no effect was found
in a variety of doses (18). The authors concluded that
serotonin did not seem to have an effect in that model.
PCPA has not been used in other chronic models of typ-
ical absence seizures. The effect PCPA on other types of
experimental seizures appears to be model dependent. For
instance, PCPA-mediated serotonin depletion prolonged
FIG. 1. Effect of para-chlorophenylalanine (PCPA) treatment on
the latency of audiogenic seizures in DBA/2J mice (28)
total slow spike-and-wave duration (SSWD) in male AY-treated and decreased the amplitude, but not frequency, of hip-
rats. PCPA treatment signiﬁcantly reduced the total SSWD dura- pocampal seizures elicited by digitoxigenin (29). Short-
tion in male AY-treated rats (n = 6) but had no effect on controls
(n = 8); vehicle had no signiﬁcant effect (n = 6). (∗∗ p < 0.01, two-
and long-term PCPA treatment decreased the severity of
tailed paired Student’s t test versus baseline). AY, AY-9944; Ctrl, seizures and delayed kindling in rats and rabbits (30, 31).
Control; Post-treat, after treatment. Similarly, PCPA delayed amygdala kindling in rabbits but
increased the severity of pentyelentetrazol-induced con-
vulsions in rats (32,33). Alternatively, PCPA increased
Brain serotonin levels
the severity of seizures induced by repetitive stimulations
Levels of 5-HIAA differed significantly within each
to the dorsal hippocampus in rat (34).
region examined (Fig. 2A). 5-HIAA levels were signif-
In our study, we confirmed that PCPA depleted the brain
icantly elevated in AY rats treated with vehicle as com-
of 5-HIAA (94–99%) and 5-HT (92–95%). Such a pro-
pared with na¨ve rats in the brainstem (51% increase, p
found reduction of serotonin in the PCPA-treated AY rat
< 0.05, Student’s t test). PCPA treatment significantly re-
is important because it demonstrates that even with a near-
duced 5-HIAA levels by 94–99% in all brain regions of
complete depletion of brain serotonin, seizures were still
control-PCPA rats when compared with na¨ve rats and in
apparent, albeit significantly reduced, in the AY model.
AY-PCPA rats when compared with AY-vehicle.
Because thalamocortical hippocampal pathways are in-
Figure 2B shows the levels of 5-HT in the brain re-
volved in the AY model (7,10), our data comport with the
gions studied. With ANOVA, significant differences were
findings of Jakala et al. (35), who reported that thalamo-
obtained in all brain regions examined. Specific compar-
cortical oscillations (36) were still apparent after PCPA-
isons showed that AY-vehicle rats had significantly ele-
mediated serotonin depletion. Similarly, PCPA had no ef-
vated 5-HT levels in both the thalamus (50% increase, p
fect on the GAERS rat model of typical absence seizures
< 0.05, Student’s t test) and brainstem (61% increase, p <
(18). These data suggest that serotonin depletion may not
0.05, Student’s t test). PCPA significantly depleted 5-HT
affect thalamocortical oscillatory activity. The ability of
levels by 92–95% in all brain regions studied when com-
PCPA to reduce SSWDs in the AY model but not SWDs
paring control-PCPA with naive rats, and AY-PCPA with
in the GAERS may lie in the inherent differences between
these two models of absence seizures. Because the hip-
As Fig. 3 shows, AY-treated rats given vehicle had sig-
pocampus displays seizure activity in the AY model (7)
nificantly reduced turnover rates in the thalamus (21%,
but not in the GAERS model (8,18), it could be hypoth-
p < 0.05, Student’s t test) and cortex (19%, p < 0.05,
esized that the hippocampus may play a role in modulat-
Student’s t test) as compared with male naive rats.
ing seizures in the AY model (37). For example, Bertram
and Zhang (38) showed strong and possibly monosynap-
tic connections between the thalamus and hippocampus.
These data indicate that PCPA-mediated serotonin de- Thus it is conceivable that serotonin depletion may act on
pletion protects against experimental AASs induced by the hippocampus to block absence seizure generation or
AY in Long Evans hooded rats. Serotonin depletion with propagation.
PCPA did not change the spontaneous and minimal spike- The brain levels of 5-HT were significantly increased
wave–like bursts duration seen in control rats. Biochemi- in the thalamus and brainstem, and the levels of 5-HIAA
cal data confirmed that PCPA caused a marked depletion were significantly increased in the thalamus of AY rats
of both 5-HT and 5-HIAA in all brain regions studied in ı
treated with vehicle compared with na¨ve rats and less so
both control and AY-treated rats. Furthermore, with the in other brain regions. The lack of significant changes in
HPLC, we demonstrated that the AY developmental treat- the cortex may be attributed to a lack of sensitivity to small
ment produced an increase in 5-HT and 5-HIAA levels changes after AY treatment. The small effects are ampli-
in some of the brain regions studied when compared with fied when serotonin turnover is calculated and significance
those in naive rats. A concomitant significant decrease was is reached. The inability of hippocampal transmitter levels
Epilepsia, Vol. 47, No. 2, 2006
244 E. BERCOVICI ET AL.
FIG. 2. Monoamine measurements from four separate brain regions: levels of 5-hydroxyindoleacetic acid (5-HIAA) and serotonin (5-HT).
A: 5-HIAA levels are elevated in male AY-treated rats as compared with naive male rats. Treatment with para-chlorophenylalanine (PCPA)
depleted 5-HIAA levels in AY and control rats as compared with their respective controls. B: 5-HT levels were markedly depleted in AY and
control rats as compared with their respective controls. AY rats showed higher 5-HT levels in the thalamus and brainstem as compared
with naive; rats (∗ p < 0.05, two-tailed unpaired Student’s t test versus male naive; ∗∗ p < 0.01, two-tailed unpaired Student’s t test versus
male naive; #p < 0.01, two-tailed unpaired Student’s t test versus AY vehicle). NB: AY-PCPA cortex 5-HIAA value is correctly labeled but
value is too small to produce noticeable bar.
to reach significance suggests that AY treatment may (32). In our study, we showed that AY rats had lower 5-
not produce as profound effects in this region and per- HIAA/5-HT ratios when compared with naive rats, which
haps warrants further investigation as to how serotonin suggests that the alterations in brain 5-HT and 5-HIAA
may modulate AY-induced seizures via the hippocampal– levels could be due to reduced serotonergic metabolism
thalamocortical circuitry. Overall, these transmitter data in this model. In the PTZ seizure model, Szyndler et al.
suggest that serotonin levels may be altered during AY- (28) demonstrated reduced serotonin-turnover rate in the
induced epileptogenesis. Alternatively, these data may hippocampus and prefrontal cortex. Concomitantly, in an-
indicate that serotonin levels are increased in response other study, Szyndler et al. (39) demonstrated that PTZ-
to seizures, possibly as a compensatory mechanism, as induced seizures reduced the binding of the SSRI citalo-
shown previously (27,32). pram in the hippocampus. These data suggest that, at least
The 5-HIAA/5-HT ratio has been used as a tool to ap- in the PTZ model, reduced serotonin uptake can be as-
proximate the serotonin-turnover ratio and metabolism sociated with the reduced turnover rate. It could be hy-
(25,26) and can be altered in response to pentylenetetra- pothesized that these compensatory mechanisms occur to
zol (PTZ) (27) as well as audiogenic seizures in mice conserve serotonin. Whether similar mechanisms explain
Epilepsia, Vol. 47, No. 2, 2006
SEROTONIN DEPLETION AND ATYPICAL ABSENCE SEIZURES 245
FIG. 3. Serotonin turnover rates (5-HIAA/5-HT) in four distinct brain regions. AY-treated rats showed reduced serotonin-turnover rates as
compared with naive rats (∗ p < 0.05, two-tailed unpaired Student’s t test vs. male naive).
the reduced serotonin-turnover rate in the AY model re- cally, the authors reported that GluR2, which controls the
mains to be elucidated. influx of Ca2+ ions, was significantly increased, thus lim-
We found that serotonin-turnover rates in the hippocam- iting neuronal Ca2+ influx. Alternatively, Bidzinski et al.
pus or the brainstem were not significantly altered in (44) found that PCPA treatment was associated with a de-
AY-vehicle rats as compared with naive rats. In the hip- crease in [3 H]muscimol binding to the GABAA receptor
pocampus, these differences may be attributed to tissue in the hippocampus (CA3 and dentate), entorhinal cortex,
variability, because both 5-HT and 5-HIAA were slightly occipital cortex, and geniculate nucleus. These data raise
elevated in AY-vehicle rats as compared with naive rats. the possibility that serotonin depletion also may modulate
This suggests that at least some degree of perturbation the GABAergic function within the thalamocortical cir-
of the serotonergic system exists. In the brainstem, 5-HT cuitry with a resultant effect on SSWDs (35,45). Further
and 5-HIAA were significantly elevated, but the overall studies are needed to investigate the possible effects of
ratio was unaltered. Because compensatory mechanisms PCPA on the GABA and glutamate systems and their con-
are likely to occur at the extremities of serotonergic neu- tribution to the attenuation of the developmentally induced
rons, it is likely that these changes would not be observed AY-SSWDs. However, in the meantime, we will consider
in the brainstem where the serotonergic cell bodies are studying the effect of enhancing serotonergic tone via se-
located. lective serotonin reuptake inhibitors (SSRIs) (17), in the
It is conceivable that PCPA could attenuate AY-induced AY-9944 model.
AASs by other mechanisms derived from serotonin deple-
tion. PCPA-mediated serotonin depletion has been shown Acknowledgment: This study was supported in part by the
Hospital for Sick Children (HSC) Paediatric Consultants, On-
to induce insomnia in cats (40). It is possible that the rats tario Graduate Scholarship (OGS), Natural Sciences and En-
in our study became more alert after serotonin depletion. gineering Research Council (NSERC), HSC Research Train-
Thus reduction of seizures could be attributed to an in- ing Competition, and Canadian Institutes of Health Research
crease in wakefulness. However, studies investigating in- (CIHR). We are grateful to Dr. McIntyre Burnham, Dick Liu, and
somnia usually do so by using cats given single high doses the technical staff at the HSC Laboratory Animal Services for the
care taken during the experiments and to Marilyn McLaughlin
of PCPA (500 mg/kg) (41). Rats seem to show more resis- for her assistance in preparation of the manuscript.
tance to insomnia induced by PCPA, and they show a rapid
rebound (42). Our dosing schedule would also minimize
the amount of insomnia produced because the drug is de- REFERENCES
livered over a 3-day period. Nevertheless, changes in the 1. Bare MA, Glauser TA, Strawsburg RH. Need for electroencephalo-
sleep–wake cycle could in part contribute to the reduced gram video confirmation of atypical absence seizures in children
seizure effects that we observed. with Lennox-Gastaut syndrome. J Child Neurol 1998;13:498–
The actions of PCPA also can be explained by assess- 2. Carmant L, Kramer U, Holmes GL, et al. Differential diagnosis
ing long-term neurochemical changes in the rat brain after of staring spells in children: a video-EEG study. Pediatr Neurol
serotonin depletion. For example, PCPA-mediated sero- 1996;14:199–202.
3. Holmes GL, McKeever M, Adamson M. Absence seizures in
tonin depletion has been shown to be associated with children: clinical and electroencephalographic features. Ann Neu-
changes in glutamate-receptor expression (43). Specifi- rol 1987;21:268–273.
Epilepsia, Vol. 47, No. 2, 2006
246 E. BERCOVICI ET AL.
4. Fernandez RJ, Samuels MA. Epilepsy. In: Samuels MA, ed. Man- fects of 5,7-dihydroxytryptamine and p-chlorophenylalanine. Neu-
ual of Neurology: Diagnosis and Therapy, 4th ed. Boston: Little, ropsychopharmacology 2001;24:399–409.
Brown, 1991:82–118. 25. Esteban S, Nicolaus C, Garmundi A, et al. Effect of orally adminis-
5. Johannessen SI, Gram L, Sillanp¨ a M, Tomson T. Intractable
a¨ tered L-tryptophan on serotonin, melatonin, and the innate immune
Epilepsy. Petersfield, U.K: Wrightson Biomedical Publishing, response in the rat. Mol Cell Biochem 2004;267:39–46.
1995. 26. Arshaduddin M, Al Kadasah S, Biary N, et al. Citalopram, a se-
6. Nolan M, Bergazer M, Chu B, et al. The clinical and neurophysio- lective serotonin reuptake inhibitor augments harmaline-induced
logical spectrum associated with atypical absence seizures. J Child tremor in rats. Behav Brain Res 2004;153:15–20.
Neurol 2005;20:404–410. 27. Szyndler J, Rok P, Maciejak P, et al. Effects of pentylenetetrazol-
7. Cortez MA, McKerlie C, Snead OC 3rd. A model of chronic atypi- induced kindling of seizures on rat emotional behavior and
cal absence seizures: EEG, pharmacology and developmental char- brain monoaminergic systems. Pharmacol Biochem Behav
acterization. Neurology 2001;56:341–349. 2002;73:851–861.
8. Snead OC 3rd, Depaulis A, Vergnes M, et al. Absence epilepsy: ad- 28. Peters RI, Lack DB. Audiogenic seizures and brain sero-
vances in experimental animal models. Adv Neurol 1999;79:253– tonin after L-tryptophan and p-chlorophenylalanine. Exp Neurol
9. Getova D, Bowery NG, Spassov V. Effects of GABAB receptor 29. Vocci FJ, Buterbaugh GG. Hippocampal seizure discharge pro-
antagonists on learning and memory retention in a rat model of duced by systemic digitoxigenin is antagonized by reserpine or
absence epilepsy. Eur J Pharmacol 1997;320:9–13. p-chlorophenylalanine. Neuropharmacology 1982;21:95–98.
10. Chan KF, Jia Z, Murphy PA, et al. Learning and memory impair- 30. Stach R, Lazarova MB, Kacz D. Serotonergic mechanism in
ment in rats with chronic atypical absence seizures. Exp Neurol seizures kindled from the rabbit amygdala. Naunyn Schmiedebergs
2004;190:328–336. Arch Pharmacol 1981;316:56–58.
11. Persad V, Cortez MA, Snead OC 3rd. A chronic model of atypical 31. Racine R, Coscina DV. Effects of midbrain raphe lesions or
absence seizures: studies of developmental and gender sensitivity. systemic p-chlorophenylalanine on the development of kindled
Epilepsy Res 2002;48:111–119. seizures in rats. Brain Res Bull 1979;4:1–7.
12. Cortez MA, Cunnane SC, Snead OC 3rd. Brain sterols in the AY- 32. Kostowski W, Bidzinski A, Hauptmann M, et al. Brain serotonin
9944 rat model of atypical absence seizures. Epilepsia 2002;43:3– and epileptic seizures in mice: a pharmacological and biochemical
8. study. Pol J Pharmacol Pharm 1978;30:41–47.
13. Cortez MA, Snead OC 3rd. Continuous spike-and-wave during 33. Lazarova M, Roussinov K, Yanev S, et al. Effect of chronic para-
slow wave sleep (CSWSS) in the AY-9944 model. Epilepsia chlorophenylalanine treatment on convulsive-seizure reactions.
2000;41(suppl 7) :243. Acta Biol Med Ger 1981;40:309–316.
14. Nielsen CK. Head and whole-body jerking in guinea pigs are differ- 34. Cavalheiro EA, Elisabetsky E, Campos CJ. Effect of brain sero-
entially modulated by 5-HT1A, 5-HT1B/1D and 5-HT2A receptor tonin level on induced hippocampal paroxysmal activity in rats.
antagonists. Eur J Pharmacol 1998;361:185–190. Pharmacol Biochem Behav 1981;15:363–366.
15. Marley E, Wozniak KM. Interactions of non-selective monoamine 35. Jakala P, Bjorklund M, Riekkinen P Jr. Suppression of neocor-
oxidase inhibitors, tranylcypromine and nialamide, with inhibitors tical high-voltage spindles by nicotinic acetylcholine and 5-HT2
of 5-hydroxytryptamine, dopamine or noradrenaline re-uptake. J receptor stimulation. Eur J Pharmacol 1996;299:47–60.
Psychiatric Res 1984;18:191–203. 36. Steriade M, Amzica F. Sleep oscillations developing into seizures
16. Koe BK, Weissman A. The pharmacology of para- in corticothalamic systems. Epilepsia 2003;44:9–20.
chlorophenylalanine, a selective depletor of serotonin stores. 37. Mistry N, Cortez MA, Snead OC 3rd. Thalamic and hippocampal
Adv Pharmacol 1968;6:29–47. ibotenic acid lesions in the AY-model of atypical absence seizures.
17. Jakus R, Graf M, Juhasz G, et al. 5-HT2C receptors inhibit Can J Neurol Sci 2003;30(suppl 2) :S33.
and 5-HT1A receptors activate the generation of spike–wave dis- 38. Bertram EH, Zhang DX. Thalamic excitation of hippocampal CA1
charges in a genetic rat model of absence epilepsy. Exp Neurol neurons: a comparison with the effects of CA3 stimulation. Neu-
2003;184:964–972. roscience 1999;92:15–26.
18. Marescaux C, Vergnes M, Depaulis A. Neurotransmission in rats’ 39. Szyndler J, Wierzba-Bobrowicz T, Maciejak P, et al.
spontaneous generalized nonconvulsive epilepsy. Epilepsy Res Pentylenetetrazol-kindling of seizures selectively decreases
Suppl 1992;8:335–343. [3H]-citalopram binding in the CA-3 area of rat hippocampus.
19. Coenen AM, Van Luijtelaar EL. Genetic animal models for absence Neurosci Lett 2002;335:49–53.
epilepsy: a review of the WAG/Rij strain of rats. Behav Genet 40. Jouvet M. Sleep and serotonin: an unfinished story. Neuropsy-
2003;33:635–655. chopharmacology 1999;21(suppl 2) :24–27.
20. Costa E, Gessa GL, Sandler M. Serotonin: New Vistas. New York: 41. Pujol JF, Buguet A, Froment JL, et al. The central metabolism
Raven Press, 1974. of serotonin in the cat during insomnia: a neurophysiological and
21. Loscher W, Fiedler M. The role of technical, biological and biochemical study after administration of p-chlorophenylalanine
pharmacological factors in the laboratory evaluation of anticon- or destruction of the raphe system. Brain Res 1971;29:195–212.
vulsant drugs, VI: seasonal influences on maximal electroshock 42. Borbely AA, Neuhaus HU, Tobler I. Effects of p-
and pentylenetetrazol seizure threshold. Epilepsy Res 1996;25:3– chlorophenylalanine and tryptophan on sleep, EEG, and
10. motor activity in the rat. Behav Brain Res 1981;2:1–22.
22. Tagliaferro P, Ramos AJ, Lopez-Costa JJ, et al. Increased nitric 43. Shutoh F, Hamada S, Shibata M, et al. Long term depletion of
oxide synthase activity in a model of serotonin depletion. Brain serotonin leads to selective changes in glutamate receptor subunits.
Res Bull 2001;54:199–205. Neurosci Res 2000;38:365–371.
23. Alvarez JC, Bothua D, Collignon I, et al. Simultaneous measure- 44. Bidzinski A, Siemiatkowski M, Czlonkoswka A, et al. The effect of
ment of dopamine, serotonin, their metabolites and tryptophan serotonin depletion on motor activity habituation, and [3H] musci-
in mouse brain homogenates by high-performance liquid chro- mol binding in the rat hippocampus. Eur J Pharmacol 1998;353:5–
matography with dual coulometric detection. Biomed Chromatogr 12.
1999;13:293–298. 45. McCormick DA. Neurotransmitter actions in the thalamus and
24. Fletcher PJ, Selhi ZF, Azampanah A, et al. Reduced brain serotonin cerebral cortex and their role in neuromodulation of thalamocorti-
activity disrupts prepulse inhibition of the acoustic startle reflex: ef- cal activity. Progr Neurobiol 1992;39:337–388.
Epilepsia, Vol. 47, No. 2, 2006