Download Amphetamine-sensitized animals show a sensorimotor gating and

Schizophrenia Research 64 (2003) 103 – 114
www.elsevier.com/locate/schres
Amphetamine-sensitized animals show a sensorimotor gating and
neurochemical abnormality similar to that of schizophrenia
Catherine C. Tenn a, Paul J. Fletcher b,c, Shitij Kapur a,c,*
a
Schizophrenia/PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, Canada
Section of Biopsychology, Centre for Addiction and Mental Health, Toronto, Ontario, Canada
c
Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada
b
Received 27 June 2002; received in revised form 6 December 2002; accepted 23 December 2002
Abstract
The aim of these studies was to examine whether amphetamine-induced sensitization in rats could be used as an animal
model to study the basis of certain abnormalities seen in schizophrenia. Specifically, these experiments examined whether rats
subjected to a sensitizing regimen of amphetamine would show the sensorimotor gating and greater amphetamine-induced
displacement of radio-raclopride binding deficit that is observed in schizophrenia. In the first experiment, animals were divided
into two groups with each rat receiving an intraperitoneal injection of amphetamine (AMPH) or saline (SAL) (1 ml/kg) three
times per week for 3 weeks for a total of nine injections. AMPH dose was increased weekly from 1 mg/kg in the first week to 3
mg/kg in the third. Twenty-two days after the last injection, prepulse inhibition (PPI) of the acoustic startle response was tested.
In addition, rats were tested for the effects of a challenge dose of 0.5 mg/kg AMPH on locomotor activity and [3H]raclopride
(RAC) binding potential (BP) in the striatum. The tests for PPI confirmed that sensorimotor gating was disrupted in the AMPHinduced sensitized-state rats at baseline. The AMPH-sensitized rats also exhibited higher locomotor response to AMPH and a
lower binding of striatal [3H]raclopride when challenged with the drug. The results were replicated and even more pronounced
in rats that were treated with AMPH for 5 weeks, with doses ranging from 1mg/kg in the first week to 5 mg/kg in the fifth.
These sensorimotor gating deficits and neurochemical (greater AMPH-induced displacement of radio-raclopride binding)
abnormalities show similarities with the pathophysiology of schizophrenia and suggest that the AMPH-sensitized-state rats
could be used to model certain aspects of schizophrenia.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Amphetamine; Schizophrenia; Sensitization; Dopamine; Prepulse inhibition; Raclopride
* Corresponding author. Schizophrenia/PET Centre, Clark
Institute, Centre for Addiction and Mental Health, 250 College
Street, Toronto, Ontario, Canada M5T 1R8. Tel.: +1-416-5358501x4361; fax: +1-416-260-4164.
E-mail address: [email protected] (S. Kapur).
Schizophrenia is a complex neuropsychiatric disorder with psychotic, cognitive and affective features.
Animal models are critical in pursuit of further knowledge about schizophrenia and in generating better
therapeutic options. Since the etiology/ies of schizophrenia is not determined, it is not possible as yet to
have an etiologically driven model. However, the
development of models that demonstrate behavioural
and neurochemical phenotypes analogous to that seen
0920-9964/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0920-9964(03)00009-4
104
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
in schizophrenia can lead to a much better insight into
the disorder.
The most common approach to modelling dopaminergic pathophysiology in schizophrenia has been the
acute challenge of a dopamine-releasing (e.g. amphetamine) or dopamine receptor stimulating (e.g. apomorphine) agent, and examining its effects on motor
behaviour (see review by Robinson and Becker, 1986;
Vanderschuren et al., 1999), sensorimotor gating
(Braff and Geyer, 1990; Mansbach et al., 1988;
Swerdlow et al., 2000) or learned associations (Moser
et al., 2000; Broersen et al., 1999). This approach has
several limitations. The hyperdopaminergic state of
schizophrenia is a chronic and self-sustaining one, not
an acute and exogenous one. In that regard, these
acute challenges in normal rats may be much more
akin to episodic drug abuse than they are to the
condition of schizophrenia. In addition, the behavioural aberration is not reflected in the baseline state but
in the challenged state of the animal. This is at odds
with schizophrenia where the patients show abnormalities both at baseline and in response to challenges
(Abi-Dargham et al., 1998, 2000; Laruelle, 2000;
Seeman and Kapur, 2000). Finally, since these challenges create their behavioural endpoints via direct
stimulation of the dopamine system, any drug that
blocks the dopamine system, particularly the dopamine D2 system is effective in these models. As a
result, the model tends to be self-referential and is thus
unlikely to reveal truly novel methods of attenuating a
hyperdopaminergic response. Thus, it is important to
work with a model of hyperdopaminergia which is
chronic, self-sustaining, does not require the contemporaneous drug administration and results in behavioural aberrations similar to schizophrenia in the
baseline state of the animal.
Much progress has been made in understanding
the sensorimotor gating deficits in patients with
schizophrenia as measured by prepulse inhibition
(PPI) of acoustic startle. PPI is the reduction of a
startle response to a high-intensity stimulus that is
preceded by a low-intensity prestimulus (Hoffman
and Ison, 1980; Braff and Geyer, 1990). PPI is
particularly valuable since it is possible to test animal
models in methods very similar to that used in
humans. Since disruption in PPI is seen in medicated
patients as well as at baseline in schizophrenics (Braff
et al., 2001), it is perhaps a trait abnormality, which
should also be present at baseline in any proposed
model of schizophrenia.
At a neurochemical level, neuroimaging data
show increased striatal dopamine transmission following amphetamine challenge in patients with
schizophrenia. Reduction in the binding potential
(BP) of [123I]IBZM or [11C]raclopride indicated an
increase in dopamine D2 receptor occupancy induced
by amphetamine. As compared to healthy control
subjects, schizophrenia patients showed a significant
increase in amphetamine-induced reduction of the
binding of the radiotracers (Abi-Dargham et al.,
1998; Brier et al., 1997; Laruelle et al., 1996). Furthermore, an increase in the BP of [123I]IBZM was
observed following depletion of endogenous dopamine with alpha-methyl-para-tyrosine in schizophrenics (Abi-Dargham et al., 2000). These results
suggest that a greater portion of D2 receptors in
schizophrenics are occupied by dopamine as compared
to their controls—both at baseline and after a challenge
of a dopamine-releasing agent (Abi-Dargham et al.,
1998; Seeman and Kapur, 2000). Thus, an animal
model of this illness should try and replicate this
endophenotype.
It is well known that repeated exposure to stimulant drugs such as amphetamine induces behavioural
and neurochemical sensitization in animals (Robinson
and Becker, 1986). The phenomenon is of interest
since amphetamine-induced psychosis resembles paranoid schizophrenia and it has been suggested that
this model may have relevance for studying neurochemical and behavioural abnormalities associated
with the psychotic aspects of schizophrenia (Robinson
and Becker, 1986). Sensitization as defined by
enhanced release of dopamine and behavioural activation in response to a drug or other challenge is, as is
schizophrenia, a chronic and enduring state.
In examining the literature on psychostimulantinduced sensitization, there are a wide variety of
regimens (number of injections, dose of drug, length
of withdrawal period, etc.) that have been utilized.
Although almost all the regimens show a behavioural
sensitization, there is variance in the degree to which
the dopaminergic system is hyperresponsive (Segal
and Kuczenski, 1992; Kolta et al., 1985; Patrick et al.,
1991; Kalivas and Stewart, 1991; Paulson and Robinson, 1995). Robinson et al. (1988) suggest that
using gradually escalating intermittent doses of
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
amphetamine and separating the induction phase from
challenge phase with a long period of withdrawal may
lead to a more intense and robust sensitized state.
Thus, we were interested in utilizing the escalatingdosing sensitization model since Robinson et al.
(1988) had reported less individual variation as well
as an increase in striatal dopamine release in animals
sensitized by this regimen.
The aim of these studies was to examine whether
rats that were sensitized by utilizing an escalating
dose of AMPH regimen would show a sensorimotor
gating (disruption of PPI) and neurochemical
(greater amphetamine-induced displacement of
radio-raclopride binding) abnormality similar to that
of schizophrenia.
1. Methods and materials
105
given 10 min to acclimatize to the equipment that had
a 65-dB white noise in the background. After this
period, subjects were presented with a series of five
startle pulse-alone trials to control for habituation of
the startle response. This series of stimuli was followed by 64 randomized trials consisting of no pulse
(0 dB), a startle pulse (110 dB, 40 ms) or three
prepulse intensities (70, 75 and 80 dB, 20 ms)
presented alone or 100 ms preceding a startle pulse.
Finally, another series of five startle pulse-alone trials
were presented. The time between trials ranged from
10 to 20 s. The startle response was measured every 1
ms for a 100-ms period from the onset of the startle
stimulus. The percent prepulse inhibition (PPI) was
calculated by the following formula: PPI = 100
( P + S/S)*100. Where P + S is the mean response
amplitude for prepulse plus startle pulse trials and S
is the mean response amplitude for the startle pulsealone trials.
1.1. Animals
1.4. Amphetamine treatment—Experiment IA
Adult male Sprague –Dawley rats, weighing 200–
225 g at the start of the experiment were used. They
were housed two per cage with free access to food and
water. The housing room was maintained at a constant
temperature of 20 F 2 jC on a 12:12 reverse light/
dark cycle. Lights were off at 8:00 AM.
1.2. Locomotor activity testing
The activity boxes were similar to the home cages
but were equipped with a row of six photocell beams
placed 3 cm above the floor of the cage. A computer
that detects the disruption of the photocell beams
recorded the number of beam breaks. Prior to beginning any study the animals were allowed to habituate
to the activity boxes 1 week before the drug treatment
and during the last 2 weeks of the withdrawal phase.
There were two habituation sessions before drug treatment and three sessions during withdrawal. The rats
were placed in the activity boxes for a period of 2 h.
1.3. Prepulse inhibition
Startle responses were assessed using SR-Lab
Startle Response systems (San Diego Instruments,
San Diego, CA, USA) as described by Sills (1999).
The rats were placed in the startle apparatus and
This experiment investigated the effects of a sensitizing regimen of amphetamine given over 3 weeks on
basal PPI of the acoustic startle reflex, and amphetamine stimulated locomotor activity.
Sixteen animals were randomly divided into two
groups with each rat receiving an intraperitoneal
injection of either D-amphetamine sulphate (AMPH;
Sigma-RBI) or 0.9% saline (SAL; 1 ml/kg). Since the
animals were housed in pairs, cage mates received the
same drug treatment. Injections were given on Monday, Wednesday and Friday for 3 weeks. AMPHpretreated rats received a total of nine AMPH injections over 3 weeks. The dose ranged from 1 to 3 mg/
kg with an increase of 1 mg/kg each week. Immediately after the injection each rat was returned to its
home cage. Following the last injection, the animals
were left drug-free for 22 days. The rats were then
tested for startle responses and PPI according to the
protocol outlined above. The startle and PPI data of
one rat (saline-pretreated) were not included since the
startle response was very high at all prepulse stimulus
intensities tested. The following day, the test for
locomotor activity was carried out. Animals were
allowed to habituate to the activity boxes for 30
min. Following this period, each rat was injected with
saline intraperitoneally and the activity was recorded
106
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
for 1 h. The animals were then injected with 0.5 mg/
kg of AMPH intraperitoneally and the activity was
measured for an additional 1 h.
1.5. [3H]raclopride binding potential in striatum—
Experiment IB
This experiment investigated the effects of an
acute injection of AMPH on [3H]raclopride displacement in AMPH-sensitized rats and their controls.
Twenty-four rats were divided into four groups of
six. Two groups of rats underwent the same AMPH
dosing regimen as the animals in Experiment 1A.
The remaining two groups were injected with saline.
Twenty-eight days after the last injection, the animals
that were AMPH-preexposed received either a SAL
or an AMPH (3 mg/kg) challenge. The SAL-preexposed rats were also divided into a SAL- or AMPHchallenged group. Twenty minutes after this injection, the animals were given a tail vein injection of
7.5 ACi [3H]raclopride (RAC) in 0.4 ml of saline.
Thirty minutes after the RAC injection, the animals
were sacrificed and the brains quickly dissected.
Specific (striatum and nucleus accumbens) and nonspecific (cerebellum) binding was determined by
scintillation counting. The ratio of specific/nonspecific provided an estimate of the D2 receptor (BP)
available. The percent occupancy of D2 receptors was
calculated by the following equation: % Occupancy =
100 (BPdrug/BPcontrol*100).
rats were divided into four groups of six. The animals
that were AMPH-preexposed received either SAL or
AMPH (3 mg/kg) challenge. The SAL-preexposed
rats were also divided into a SAL- or AMPH-challenged group. Twenty minutes after this injection, the
animals were given a tail vein injection of 7.5 ACi
[3H]raclopride in 0.4 ml of saline. Thirty minutes after
the RAC injection, the animals were sacrificed and the
brains quickly dissected. Specific (striatum and
nucleus accumbens) and nonspecific (cerebellum)
binding was determined by scintillation counting.
The percent occupancy of D2 receptors was calculated
as described above in Experiment 1B.
1.7. Experiment III
1.7.1. Confirmation and duration of PPI abnormality
In the third set of experiments, 32 animals were
randomly divided into two groups with each rat
receiving an intraperitoneal injection of amphetamine
or 0.9% saline (1 ml/kg). Injections were given on
Monday, Wednesday and Friday for 3 weeks. AMPHpretreated rats received increasing doses of AMPH for
a total of nine injections over 3 weeks. The dose
ranged from 1 to 3 mg/kg with an increase of 1 mg/kg
each week. Immediately after the injection each rat
was returned to its home cage. The rats were tested for
startle responses and PPI according to the protocol
outlined above. The effect of pretreatment on basal
PPI of startle reflex was examined on the 21st, 40th
and 60th day after the sensitization regimen.
1.6. Amphetamine treatment—Experiment II
In the second set of experiments, 24 rats were used.
The AMPH-treated rats were given injections on
Monday, Wednesday and Friday for 5 weeks.
AMPH-pretreated rats received increasing doses of
AMPH for a total of 15 injections over 5 weeks. The
dose ranged from 1 to 5 mg/kg with an increase of 1
mg/kg each week. SAL-pretreated rats received injections of saline. Immediately after the injection each rat
was returned to its home cage. Following the last
injection, the animals were left drug-free for several
weeks. Basal PPI of the acoustic startle reflex was
examined in these rats after 22 days of withdrawal.
The animals were then left alone for 1 week before
they were used to determine the effects of amphetamine-induced displacement of RAC binding. The 24
2. Results
2.1. Experiment IA
2.1.1. Locomotor activity
Animals used in this experiment were preexposed
to amphetamine or saline for 3 weeks and tested 22
days after the last injection for their locomotor
response to saline and a challenge of 0.5 mg/kg of
amphetamine (Fig. 1A). There was no difference
between the amphetamine- and saline-pretreated
groups during the 30-min habituation period. However, there was a significant difference in total activity
counts between the two groups during the 1 h following the saline injection (t = 3.0, df = 14, p < 0.01, t-test)
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
107
Fig. 1. (A) Animals were preexposed to either saline (Sal) or amphetamine (Amph) for 3 weeks and were tested 24 days after sensitization
injection. Locomotor activity was measured during a 30-min period (habituation, Habit), a 1-h period following SAL injection and a 1-h
period following an AMPH challenge (0.5 mg/kg). Values are means F S.E.M. for eight rats in each group. (B) Analyses of the total activity
counts (from A) showed there was a significant difference between the saline- (black bars) and amphetamine-pretreated (grey bars) animals
after a saline (*p < 0.01, t-test) or an amphetamine challenge (**p < 0.001, t-test). The values are means F S.E.M. presented for eight rats per
group.
and during the 1 h following the amphetamine challenge (t = 10.2, df = 14, p < 0.001, t-test) (Fig. 1B).
Thus, amphetamine-sensitized rats showed significantly greater locomotor stimulation in response to
injection with saline and amphetamine.
2.1.2. Prepulse inhibition of acoustic startle
Pretreatment had no effect on startle response to
pulse-alone trials (means F S.E.M. startle amplitudes
were 370.9 F 42.9 and 319.2 F 31.1 for SAL- and
AMPH-preexposed groups, respectively, [ F(1,13) =
0.984, p = 0.34]). Although there was no effect of
pretreatment on startle response to prepulse + pulse
trials, [ F(1,13) = 1.36, p < 0.26], when the data was
expressed as %PPI scores, it revealed significant
effects of pretreatment (AMPH vs. SAL) [ F(1,13) =
5.10, p = 0.042] and prepulse intensities (70, 75 and
80 dB) [ F(2,26) = 33.7, p < 0.001], with AMPH-sensitized animals showing lower %PPI. Post hoc
comparisons using Tukey’s test revealed that amphetamine pretreatment reduced PPI levels at the prepulse intensity of 70 dB ( p < 0.01) but the difference
at 75 and 80 dB prepulse intensities was not significant (Fig. 2A).
108
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
Fig. 2. Prepulse inhibition of acoustic startle in rats previously sensitized to amphetamine. The rats were tested 22 days after sensitization to
amphetamine. Animals received either saline (Sal) or amphetamine (Amph) for (A) 3 weeks or for (B) 5 weeks and tested 22 days after their last
drug or vehicle injection. Values are means F S.E.M. for 7 – 12 rats per group (**p < 0.01).
2.2. Experiment IB
2.2.1. [3H]raclopride binding potential in striatum
The [3H]raclopride binding potential (BP) was
determined in the striata of saline- and amphetaminetreated animals 28 days after the last injection. Those
animals that were saline-pretreated and challenged
with saline were used as the control group (Fig. 3A).
A two-way ANOVA was used to analyze the binding
potential. There was a main effect of pretreatment
[ F(1,20) = 36.24, p < 0.001] as well as a main effect
of the challenge [ F(1,20) = 12.40, p < 0.05]. Post hoc
comparisons using Tukey’s test showed that when
challenged with 3 mg/kg of amphetamine, saline-pretreated rats showed a decrease (12%) in BP but this
was not significantly different from controls. On the
other hand, AMPH-sensitized rats showed a significant decrease in striatal BP compared to the control
group following an acute SAL (24%; p < 0.01) or
AMPH (36%; p < 0.001) injection.
2.3. Experiment II
2.3.1. Prepulse inhibition of acoustic startle
Pretreatment (AMPH vs. SAL) had no effect on
startle response to pulse-alone trials (means FS.E.M.
startle amplitudes were 257.9 F 30.3 and 233.1 F
25.3 for SAL- and AMPH-preexposed groups,
respectively [ F(1,22) = 0.397, p = 0.53]). There was
no main effect of pretreatment on startle response to
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
109
Fig. 3. Tritiated raclopride binding potential (BP) in striatum of rats treated for (A) 3 weeks and for (B) 5 weeks. Animals were challenged with
either SAL or AMPH (3 mg/kg) 28 days after the last drug injection. Those animals that were SAL-pretreated and challenged with saline (Sal/
Sal) served as controls. SAL-pretreated rats showed a slight decrease in BP when challenged with amphetamine (Sal/Amp). AMPH-pretreated
rats showed a significant decrease in BP following a saline (Sen/Sal) or amphetamine challenge (Sen/Amp). Values are means F S.E.M. for five
to six rats per group (*p < 0.01, **p < 0.001 vs. control).
prepulse + pulse trials [ F(1,22) = 0.60, p = 0.45], but
there was a significant pretreatment prepulse stimulus intensity interaction [ F(2,44)= 4.37, p = 0.02]. A
post hoc analysis showed the interaction was being
driven by a highly significant difference between
AMPH vs. SAL at the 70-dB stimulus intensity.
When the data was expressed as %PPI scores, there
were significant effects of pretreatment [ F(1,22) =
9.42, p < 0.01] as well as pretreatment prepulse
stimulus interaction [ F(2,44) = 14.77, p < 0.001].
Post hoc comparisons using Tukey’s test revealed
that AMPH pretreatment reduced PPI levels at the
prepulse intensity of 70 dB ( p < 0.001) but the
difference at 75 dB ( p = 0.62) and 80 dB ( p = 1.0)
prepulse intensities was not significant (Fig. 2B).
2.3.2. [3H]raclopride binding potential in striatum
The [3H]raclopride binding potential in the striata
of animals treated with amphetamine for 5 weeks
showed similar results as in Experiment IB. A twoway ANOVA on the binding potential revealed a main
effect of pretreatment [ F(1,20) = 34.56, p < 0.001] as
well as a main effect of the challenge [ F(1,20) = 10.64,
p < 0.05]. Post hoc Tukey comparison indicated that
saline-pretreated rats, when challenged with 3 mg/kg
of amphetamine, showed a decrease ( f 14%) in BP
but this was not significantly different from the control
group. However, the AMPH-sensitized rats showed a
significant decrease in BP as compared to controls
after SAL (25%; p < 0.01) or AMPH injection (41%;
p < 0.001) (see Fig. 3B).
110
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
2.4. Experiment III
2.4.1. Confirmation and duration of PPI abnormality
On completion of Experiments I and II, it was
noticed that whereas PPI was markedly disrupted by
AMPH in the 5 mg/kg group (either in absolute terms
or in terms of %PPI), the effect of the drug was not
as robust in the 3 mg/kg group (where %PPI was
significantly different, but, absolute startle values did
not reach significance). We were concerned that this
reflected a lack of power in the 3 mg/kg group as
opposed to a qualitative difference. Therefore, we
repeated the same 3-week escalating dose of AMPH
regimen with a larger sample size (N = 16 vs. 8 rats
per group) and examined the effect in more detail
(i.e. not only 21, but also, after 40 and 60 days of
withdrawal).
As before, pretreatment (AMPH vs. SAL) had no
effect on startle response to pulse-alone trials on any of
the days examined: day 21 (means F S.E.M. startle
amplitudes were 244.8 F 10.6 and 240.4 F 8.1 for
SAL- and AMPH-preexposed groups, respectively
[ F(1,30) = 0.11, p = 0.75]), day 40 (means F S.E.M.
startle amplitudes were 250.9 F 11.9 and 255.1
F 13.0 for SAL- and AMPH-preexposed animals,
respectively, [ F(1,30) = 0.055, p = 0.82]) or day 60
Fig. 4. Effect of escalating dose of amphetamine on startle magnitude and prepulse inhibition of acoustic startle after various withdrawal periods.
(A) Mean F S.E.M. startle magnitude for 21, 40 and 60 days after last injection. Saline-preexposed rats received either startle pulse (Sal/P-alone)
or a prepulse stimulus followed by the startle pulse (Sal/P + 70) trials. Amphetamine-preexposed rats received either startle pulse (Amp/P-alone)
or a prepulse stimulus followed by the startle pulse (Amp/P + 70) trials. (B) Prepulse inhibition of acoustic startle in rats sensitized to
amphetamine. Animals received either saline (SAL) or amphetamine (AMPH) for 3 weeks and tested 21, 40 and 60 days after their last
injection. N = 16 rats per group (*p < 0.05, **p < 0.001).
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
(means F S.E.M. startle amplitudes were 254.5F 12.2
and 251.7 F 11.4 for SAL- and AMPH-preexposed
rats, respectively [ F(1,30) = 0.028, p = 0.87]. However, pretreatment (AMPH vs. SAL) had a significant
effect on startle response to prepulse + pulse trials,
animals sensitized with AMPH showed a higher startle
magnitude in prepulse + pulse trials on all the days
measured: day 21 [ F(1,30) = 7.45, p < 0.01]; day 40
[ F(1,30) = 4.38, p = 0.045] and day 60 [ F(1,30) = 4.30,
p = 0.047] (Fig. 4).
When the data was expressed as %PPI scores, they
confirmed the previous finding that AMPH sensitization disrupts PPI and that this was most prominent at
the 70-dB prepulse intensity. On each of the three
withdrawal durations the findings were similar. On
day 21 of the withdrawal period, there were significant
effects of pretreatment [ F(1,30) = 12.9, p = 0.001] as
well as pretreatment prepulse stimulus interaction
[ F(2,60) = 7.2, p = 0.002]. On withdrawal day 40, there
was a main effect of pretreatment [ F(1,30) = 15.6,
p < 0.001] as well as a significant pretreatment preprepulse stimulus interaction [ F(2,60) = 23.3, p <
0.001] with the difference being most prominent
at 70 dB ( p < 0.001, Tukey’s test). On withdrawal
day 60, there was a main effect of pretreatment
[ F(1,30) = 31.5, p < 0.001] as well as a pretreatment prepulse stimulus interaction [ F(2,60) =
25.7, p < 0.001]. Post hoc comparisons revealed that
AMPH pretreatment significantly reduced PPI
scores at the 70-dB level ( p < 0.001). Again, there
were no differences between pretreatment at the
other two prepulse intensities.
3. Discussion
The present study shows that amphetamine sensitizationdisruptedPPIatbaselineandthiswasaccompaniedby
a lower level of raclopride binding to dopamine D2
receptors in the sensitized animals under saline and drug
challenge.Inaddition, thesensitized state model induced
sensitizationnotjusttoachallengedoseofamphetamine,
but also, to an injection of saline—an effect that may
reflect a sensitized stress response.
Two previous studies (Druhan et al., 1998; Zhang
et al., 1998) have examined PPI disruption after
sensitizing regimens of amphetamine. In their study,
Druhan et al. (1998) found that amphetamine induced
111
sensitization of locomotor activity but did not disrupt
PPI following an amphetamine challenge. Zhang et al.
(1998), using similar drug administration procedures
but with repeated startle testing, showed sensitization
to the disruptive effect of amphetamine on PPI
although they did not report PPI in nonchallenged
rats. It should be noted that, in our study, we found a
decrease in PPI at baseline in the absence of any
challenge. In this regard, the sensitized state model, at
baseline, is more like patients with schizophrenia
(Braff et al., 1978, 2001), as both show PPI impairments without an additional challenge.
A recent report (Russig, 2001) showed a disruption
of latent inhibition during withdrawal from a repeated
escalating dose of amphetamine regimen. Latent
inhibition and PPI are often used as measures of
‘gating deficits’ although they do not represent the
same construct: latent inhibition is a model of ‘gating’
of attention through the learning of the nonsignificance of stimuli, whereas PPI is a model of ‘gating’ of
a sensorimotor reflex. However, both are reported to
be disrupted in schizophrenic patients (Braff et al.,
1992; Swerdlow et al., 1994; Gray et al., 1995) and it
is intriguing in this regard that both are also abnormal
in the amphetamine-sensitized rats.
Recently, Murphy et al. (2001) reported a disruption in latent inhibition in rats treated with an escalating dose of amphetamine. However, they did not
observe PPI disruption during any of the withdrawal
days tested. Although their sensitization procedures
are similar to ours (e.g. dosages and withdrawal
times), there were some notable differences. In our
study, AMPH injections were administered once daily
over several weeks with intermittent drug-free days
(refer to Methods and materials). In their study,
Murphy et al. (2001) had administered amphetamine
three times daily over 6 days. There were no drug-free
days during their schedule of escalating dosages.
Robinson and Becker (1986) had previously reported
that a more intense and robust sensitized state is
produced when AMPH is given intermittently. Furthermore, Robinson and Becker (1986) pointed out
that when AMPH injections are administered too
close together, this was similar to continuous administration of the drug, which could lead to neurotoxicity. In addition, we had used a different strain of
rats—Sprague – Dawley vs. Wistars. In a recent
review by Geyer et al. (2001), it was shown that there
112
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
are considerably more studies reporting the use of
Sprague – Dawley than Wistar rats for testing the
effects of amphetamine on PPI. Furthermore, a large
percentage of these studies using Sprague – Dawley
rats showed amphetamine disrupted PPI. These differences between Murphy et al. (2001) and our study
may account for the dissimilar observations.
Previously, Sills (1999) examined the effect of an
acute injection of amphetamine at various doses on
PPI. The disruptive effects of amphetamine were
evident only when the prepulse was just a few
decibels above the background noise. Although the
precise mechanism for the disruption of PPI is not
evident, it is reasonable to propose that the heightened
dopaminergic transmission associated with amphetamine-sensitized state may be contributory.
The present study examined the binding of
[3H]raclopride to striatal dopamine D2 receptors in
the presence and absence of an amphetamine challenge. These experiments were carried out in an effort
to try and replicate the abnormalities noted in patients
with schizophrenia after an amphetamine challenge,
as measured by [11C]raclopride and [123I]IBZM (Laruelle et al., 1996; Brier et al., 1997; Abi-Dargham et
al., 1998). Similar to schizophrenic subjects, the
sensitized rats showed a lower binding of striatal
[3H]raclopride compared to their controls when challenged with amphetamine. In a manner that is dissimilar from schizophrenia, the amphetamine-sensitized
rats showed a lower level of [3H]raclopride even when
challenged with saline. These findings were replicated
in two different cohorts and are likely to be reliable.
However, the precise mechanism for this finding is
not clear. The binding of [3H]raclopride in vivo
represents a dynamic competition between the tracer
amounts of the radioligand and endogenous dopamine
for the D2 receptors (Laruelle, 2000). An increased
release of dopamine in response to a challenge, a
decreased number of dopamine receptors or a change
in their affinity could, in principle, contribute to the
findings observed. Microdialysis studies have shown
that amphetamine-sensitized animals show a higher
dopamine release to amphetamine (Paulson and Robinson, 1995) as well as a nonpharmacological stressor
such as foot shock (Hamamura and Fibiger, 1993). It
is possible that the saline injection in sensitized rats
was a sufficient stressor to provoke dopamine release.
Indeed, the behavioural finding that amphetamine-
sensitized rats showed increased locomotor activity
in response to a saline injection provides some
evidence that amphetamine-sensitized rats are more
responsive than nonsensitized rats to this manipulation. Thus, in keeping with the microdialysis findings, the data is likely to represent an alteration in
dopamine release, however, the alternative explanations cannot be ruled out and we are currently
undertaking in vivo/in vitro experiments to resolve
this issue.
We are not aware of any previous efforts in rodents
to examine [11C]raclopride or other ligand binding
after amphetamine sensitization, however, Castner et
al. (2000) found that whereas low doses of amphetamine in rhesus monkeys induced behavioural changes
(e.g. fine motor and oral stereotypies, static posturing,
etc.), a decrease in the percentage of amphetamineinduced displacement of [123I]IBZM in the striatum
was observed. There are several differences between
the present study and theirs: differences in techniques
(between-group ex vivo radioligand binding vs.
within-subject SPECT neuroimaging); species
(rodents vs. monkeys); sample size, the doses of
amphetamine used and the sensitization regimen and
challenge. Rather than speculate on the precise cause
for this discrepancy, it may be more prudent to wait
until this discrepancy is replicated.
There are of course, several differences between
schizophrenia and this sensitized state animal model.
Schizophrenia is not induced by repeated psychostimulant injections, though it should be noted that
chronic amphetamine abuse does lead to a picture
quite indistinguishable from schizophrenia, exhibiting
both positive and negative symptoms (Harris and
Batki, 2000). Thus, the amphetamine-induced sensitized state model shown here is a model replicating
the pathophysiology of schizophrenia, not its etiology.
Secondly, there is an increasing consensus that the
dopaminergic abnormalities in schizophrenia are not
primary, but secondary to a prefrontal or a glutamate
dysfunction (Weinberger, 1987; Deutch, 1992; Grace,
1991). It is of interest in this regard that alterations in
excitatory amino acids, and hypodopaminergia in the
prefrontal cortex have been implicated causally in the
development of the amphetamine-induced sensitized
state (Wolf, 1998; Karler et al., 1998). As such, the
sensitized state model may provide a link between the
classical dopamine hypothesis and the more encom-
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
passing glutamate hypothesis and the prefrontal
neuro-developmental hypothesis of schizophrenia.
In summary, after discontinuing treatment with
escalating doses of amphetamine, rats in their sensitized state showed a hyperlocomotor response to
novelty, a heightened displacement of [3H]raclopride
in response to saline and amphetamine as well as a
disruption of PPI. This behavioural and neurochemical findings bear similarity to the pathophysiology of
schizophrenia. Given that these findings are likely
long-lasting and do not require acute pharmacological
challenges to maintain them, the present results
indicate that the amphetamine-sensitized state may
serve as a valuable pathophysiological model of
schizophrenia.
Acknowledgements
The authors would like to thank Karin Korth,
Barbara Brownlee, Alex Kecojevic and Suzi VanderSpek for their technical assistance, and Drs. T.L.
Sills and T.E. Robinson for their expert advice. This
work was supported in part by the Canadian
Research Chair in Schizophrenia and Therapeutic
Neuroscience, as well as operating grants from the
CIHR (Canada).
References
Abi-Dargham, A., Gil, R., Krystal, J., et al., 1998. Increased striatal
dopamine transmission in schizophrenia: conformation in a second cohort. Am. J. Psychiatry 155, 761 – 767.
Abi-Dargham, A., Rodenhiser, J., Printz, D., et al., 2000. Increased
baseline occupancy of D2 receptors by dopamine in schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 97, 8104 – 8109.
Braff, D.L., Geyer, M.A., 1990. Sensorimotor gating and schizophrenia: human and animal model studies. Arch. Gen. Psychiatry 47, 181 – 188.
Braff, D.L., Stone, C., Callaway, E., et al., 1978. Prestimulus effects
on human startle reflex in normals and schizophrenics. Psychophysiology 15, 339 – 343.
Braff, D.L., Grillion, C., Geyer, M.A., 1992. Gating and habituation
of the startle reflex in schizophrenic patients. Arch. Gen. Psychiatry 49, 206 – 215.
Braff, D.L., Geyer, M.A., Light, G.A., et al., 2001. Impact of prepulse characteristics on the detection of sensorimotor gating
deficits in schizophrenia. Schizophr. Res. 49, 171 – 178.
Brier, A., Su, T.P., Saunders, R., et al., 1997. Schizophrenia is
associated with elevated amphetamine-induced synaptic dopa-
113
mine concentrations: evidence from a novel positron emission
tomography method. Proc. Natl. Acad. Sci. U. S. A. 94,
2569 – 2574.
Broersen, L.M., Feldon, J., Weiner, I., 1999. Dissociative effects of
apomorphine infusions into the medial prefrontal cortex of rats
on latent inhibition, prepulse inhibition and amphetamine-induced locomotion. Neuroscience 94, 39 – 46.
Castner, S.A., Al-Tikriti, M.S., Baldwin, R.M., et al., 2000. Behavioural changes and [123I]IBZM equilibrium SPECT measurement of amphetamine-induced dopamine release in rhesus
monkeys exposed to subchronic amphetamine. Neuropsychopharmacology 22, 4 – 13.
Deutch, A.Y., 1992. The regulation of subcortical dopamine systems by the prefrontal cortex: interactions of central dopamine
systems and pathogenesis of schizophrenia. J. Neural Transm.,
Suppl. 36, 61 – 89.
Druhan, J.P., Geyer, M.A., Valentino, R.J., 1998. Lack of sensitization to the effects of D-amphetamine and apomorphine on sensorimotor gating in rats. Psychopharmacology 135, 296 – 304.
Geyer, M.A., Krebs-Thomson, K., Braff, D.L., Swerdlow, N.R.,
2001. Pharmacological studies of prepulse inhibition models
of sensorimotor gating deficits in schizophrenia: a decade in
review. Psychopharmacology 156, 117 – 154.
Grace, A.A., 1991. Phasic versus tonic dopamine release and the
modulation of dopamine system responsivity: a hypothesis for
the etiology of schizophrenia. Neuroscience 41, 1 – 24.
Gray, N.S., Hemsley, D.R., Gray, J.A., Kerwin, R.W., 1995. Latent
inhibition in drug naı̈ve schizophrenics: relationships to duration
of illness and dopamine D2 binding using SPET. Schizophr.
Res. 17, 95 – 107.
Hamamura, T., Fibiger, H.C., 1993. Enhanced stress-induced dopamine release in the prefrontal cortex of amphetamine-sensitized
rats. Eur. J. Pharmacol. 237, 65 – 71.
Harris, D., Batki, S.L., 2000. Stimulant psychosis: symptom profile
and acute clinical course. Am. J. Addict. 9, 28 – 37.
Hoffman, H.S., Ison, J.R., 1980. Reflex modification in the domain
of startle: I. Some empirical findings and their implications for
how the nervous system processes sensory input. Psychol. Rev.
87, 175 – 189.
Kalivas, P.W., Stewart, J., 1991. Dopamine transmission in the
initiation and expression of drug and stress induced sensitization
of motor activity. Brain Res. Rev. 16, 223 – 244.
Karler, R., Calder, L.D., Thai, D.K., Bedingfield, J.B., 1998. The
role of dopamine in the mouse frontal cortex: a new hypothesis
of behavioural sensitization to amphetamine and cocaine. Pharmacol. Biochem. Behav. 61, 435 – 443.
Kolta, M.G., Shreve, P., De Souza, V., Uretsky, N.J., 1985. Time
course of the development of the enhanced behavioural and
biochemical responses to amphetamine after pretreament with
amphetamine. Neuropharmacology 24, 823 – 829.
Laruelle, M., 2000. The role of endogenous sensitization in the
pathophysiology of schizophrenia: implications from recent
brain imaging studies. Brain Res. Rev. 31, 371 – 384.
Laruelle, M., Abi-Dargham, A., van Dyck, C.H., et al., 1996. Single
photon emission computerized tomography imaging of amphetamine-induced dopamine release in drug free schizophrenic subjects. Proc. Natl. Acad. Sci. U. S. A. 93, 9235 – 9240.
114
C.C. Tenn et al. / Schizophrenia Research 64 (2003) 103–114
Mansbach, R.S., Geyer, M.A., Braff, D.L., 1988. Dopaminergic
stimulation disrupts sensorimotor gating in the rat. Psychopharmacology 94, 507 – 514.
Moser, P.C., Hitchcock, J.M., Lister, S., Moran, P.M., 2000. The
pharmacology of latent inhibition as an animal model of schizophrenia. Brain Res. Rev. 33, 275 – 307.
Murphy, C.A., Fend, M., Russig, H., Feldon, J., 2001. Latent inhibition, but not prepulse inhibition, is reduced during withdrawal from an escalating dosage schedule of amphetamine.
Behav. Neurosci. 115, 1247 – 1256.
Patrick, S.L., Thompson, T.L., Walker, J.M., Patrick, R.L., 1991.
Concomitant sensitization of amphetamine-induced behavioural
stimulation and in vivo dopamine release from the rat caudate
nucleus. Brain Res. 538, 343 – 346.
Paulson, P.E., Robinson, T.E., 1995. Amphetamine-induced timedependent sensitization of dopamine neurotransmission in the
dorsal and ventral striatum: a microdialysis study in behaving
rats. Synapse 19, 56 – 65.
Robinson, T.E., Becker, J.B., 1986. Enduring changes in brain and
behaviour produced by chronic amphetamine administration: a
review and evaluation of animal models of amphetamine psychosis. Brain Res. Rev. 396, 157 – 198.
Robinson, T.E., Jurson, P.A., Bennett, J.A., Bentgen, K.M., 1988.
Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by past experience
with (+)-amphetamine: a microdialysis study in freely moving
rats. Brain Res. 462, 211 – 222.
Russig, H., 2001. Disrupted latent inhibition and increased fear
conditioning during withdrawal from repeated escalating dosage schedule of amphetamine: a potential new animal model for
attentional dysfunctions associated with positive symptoms of
schizophrenia. World J. Biol. Psychiatry Abstr., P003 – P013.
Seeman, P., Kapur, S., 2000. Schizophrenia: more dopamine, more
D2 receptors. Proc. Natl. Acad. Sci. U. S. A. 97, 7673 – 7675.
Segal, D.S., Kuczenski, R., 1992. In vivo microdialysis reveals a
diminished amphetamine-induced DA response corresponding
to behavioural sensitization produced by repeated amphetamine
pre-treatment. Brain Res. 571, 330 – 337.
Sills, T.L., 1999. Amphetamine dose dependently disrupts prepulse
inhibition of the acoustic startle response in rats within a narrow
time window. Brain Res. Bull. 48, 445 – 448.
Swerdlow, N.R., Braff, D.L., Taaid, N., Geyer, M.A., 1994. Assessing the validity of an animal model of sensorimotor gating
deficits in schizophrenic patients. Arch. Gen. Psychiatry 51,
139 – 154.
Swerdlow, N.R., Martinez, Z.A., Hanlon, F.M., et al., 2000. Toward
understanding the biology of a complex phenotype: rat strain
and substrain differences in the sensorimotor gating—disruptive
effects of dopamine agonists. J. Neurosci. 20, 4325 – 4336.
Vanderschuren, L.J., Schmidt, E.D., De Varies, T.J., et al., 1999. A
single exposure to amphetamine is sufficient to induce longterm behavioural, neuroendocrine, and neurochemical sensitization in rats. J. Neurosci. 19, 9579 – 9586.
Weinberger, D.R., 1987. Implications of normal brain development
for the pathogenesis of schizophrenia. Arch. Gen. Psychiatry 44,
660 – 669.
Wolf, M.E., 1998. The role of excitatory amino acids in behavioural
sensitization to psychomotor stimulants. Progress. Neurobiol.
54, 679 – 720.
Zhang, J., Engel, J.A., Soderpalm, B., Svensson, L., 1998. Repeated administration of amphetamine induces sensitization to
its disruptive effect on prepulse inhibition in the rat. Psychopharmacology 135, 401 – 406.