Serotonin Depletion Attenuates AY-9944–Mediated Atypical .pdf

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					Epilepsia, 47(2):240–246, 2006
Blackwell Publishing, Inc.
C 2006 International League Against Epilepsy

      Serotonin Depletion Attenuates AY-9944–Mediated Atypical
                          Absence Seizures

          ∗ †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                                                        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
                                                               AY model
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
                                                               HPLC analysis
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
AY-vehicle–treated rats.
                                                                        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
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