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The Journal of Neuroscience, January 1, 2001, 21(1):305-313
Prepulse Inhibition Deficits and Perseverative Motor Patterns in
Dopamine Transporter Knock-Out Mice: Differential Effects of D1
and D2 Receptor Antagonists
Rebecca J.
Ralph1,
Martin P.
Paulus2,
Fabio
Fumagalli3,
Marc G.
Caron4, and
Mark A.
Geyer2
Departments of 1 Neuroscience and
2 Psychiatry, University of California San Diego, La Jolla,
California 92093-0804, 3 Center of Neuropharmacology,
Institute of Pharmacological Sciences, University of Milan, 20133 Milan, Italy, and 4 Howard Hughes Medical Institute,
Department of Cell Biology, Duke University Medical Center, Durham,
North Carolina 27710
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ABSTRACT |
Dopamine is known to regulate several behavioral phenomena,
including sensorimotor gating and aspects of motor activity. The roles
of dopamine D1 and D2 receptors in these behaviors have been documented
in the rat literature, but few reports exist on their role in mice. We
used dopamine transporter (DAT) ( /) mice to examine the behavioral
consequences of a chronically hyperdopaminergic state, challenging them
with the preferential dopamine D2 receptor antagonist raclopride and D1
receptor antagonist SCH23390. At baseline, DAT (/) mice exhibited
deficient sensorimotor gating as measured by prepulse inhibition (PPI)
of the startle response, exhibited nonfocal perseverative patterns of
locomotion, and were hyperactive in a novel environment. Pretreatment
with raclopride significantly increased PPI in the DAT (/) mice,
whereas SCH23390 had no significant effect. Blockade of D2 receptors
did not affect the predominantly straight patterns of motor behavior
produced by the DAT (/) mice, but antagonism of D1 receptors
significantly attenuated the perseverative patterns, producing more of
a meandering behavior seen in the DAT (+/+) control mice. Both D1 and
D2 receptor antagonists decreased the hyperactivity seen in the DAT
(/) mice. These findings support the role of the D2, but not the
D1, receptor in the modulation of PPI in mice. Furthermore, D1 receptor activation appears to be the critical substrate for the expression of
perseverative patterns of motor behavior, whereas both D1 and D2
receptors appear to regulate the amount of motor activity.
Key words:
dopamine; prepulse inhibition; mice; behavior; dopamine
transporter; locomotor activity; perseveration; D1 receptor; D2
receptor
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INTRODUCTION |
Prepulse inhibition (PPI) of the
startle response, an operational measure of sensorimotor gating, is a
cross-species phenomenon in which the startle response is reduced when
the startling stimulus is preceded by a low-intensity prepulse (Graham,
1975 ; Hoffman and Ison, 1980). Disruptions in PPI are produced in
rodents using dopamine (DA) agonists such as amphetamine or apomorphine
(Mansbach et al., 1988; Swerdlow et al., 1991). The DA D2 receptor
appears to be a key modulator of these DA-stimulated disruptions of PPI because antagonism of D2 receptors will reverse an apomorphine-induced disruption of PPI (Mansbach et al., 1988; Swerdlow et al., 1991; Wan et
al., 1996), whereas direct stimulation of D2 receptors produces
disruptions in PPI (Peng et al., 1990; Wan et al., 1996). A more
limited role of the D1 receptor has been implicated in the modulation
of PPI, in which a synergistic relationship between D1 and D2 receptors
is thought to exist (Peng et al., 1990; Wan et al., 1996). In studies
using mice, a mixed D1/D2 agonist disrupts PPI (Dulawa and Geyer, 1996;
Curzon and Decker, 1998), but the specific role of each receptor
subtype remains unclear. Using genetically altered mice, we have shown
that the D2 receptor is necessary for amphetamine to exert its
disruptive effects on PPI (Ralph et al., 1999), but the role of the
D1 receptor in PPI is yet to be examined in mice.
Along with modulating aspects of sensorimotor gating, DA has a
demonstrated role in the expression of motor behavior. Both the D1 and
D2 receptor systems are thought to regulate some of these behavioral
processes because experiments have shown that antagonists of both
systems will attenuate amphetamine- or cocaine-induced hyperactivity in
rats and mice (Paulus and Geyer, 1991a; O'Neill and Shaw, 1999).
Changes in the patterns of motor behavior have also been reported using
DA agonists in which the direct D1/D2 agonist apomorphine produced
perseverative motor patterns (Geyer et al., 1986, 1987; Paulus and
Geyer, 1991b). Furthermore, D1, but not D2, receptor antagonism blocked
perseverative locomotor behavior induced by amphetamine (Fritts et al.,
1997). These findings suggest that there is a dissociation in the
contributions of D1 and D2 receptors in the perseverative aspect of
motor behavior that is produced by DA activation in which D2 receptors
appear to control the amount, whereas D1 seems to influence the quality of the motor pattern.
Using gene-deletion technology, a dopamine transporter (DAT) null
mutant mouse has been created, providing a unique opportunity to study
the behavioral consequences of a chronically dysregulated DA system. We
hypothesized that the DAT (/) mice, with their hyperdopaminergic
tone, would have disrupted PPI and would be perseverative and
hyperactive in their motor behavior. We report here on two separate DAT
cohorts to confirm these behavioral phenotypes. Furthermore, we
investigated the differential role of the DA D1 and D2 receptor
subtypes in the modulation of these two behavioral phenotypes in the
DAT mice.
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MATERIALS AND METHODS |
Subjects. The DAT mutant mice [cohort 1: 23 (+/+),
47 (+/), and 9 (/) male and female mice; cohort 2: female, 20 (+/+), 22 (+/), and 17 (/); male, 18 (+/+), 19 (+/), and 18 (/)] were generated at University of California San Diego in an
Association for Assessment and Accreditation of Laboratory Animal
Care-approved animal facility using parental (+/) mice from Duke
University (Giros et al., 1996). The animal facility meets all federal
and state requirements for animal care, and federal and state
guidelines for the care and treatment of laboratory animals are
followed. M.G.C. and R.J.R. performed the genotyping of the mice. Mouse pups were weaned at 4 weeks of age and were group-housed (segregated by
sex) in a climate-controlled animal colony with a reversed day/night
cycle (lights on at 7:00 P.M., off at 7:00 A.M.). All behavioral
testing started at ~8-9 weeks of age and occurred between 9:00 A.M.
and 5:00 P.M. Food (Harlan Teklab, Madison, WI) and water were
available throughout the experiments, except during behavioral testing.
Drugs. Raclopride HCl and SCH23390 were obtained from
RBI/Sigma (St. Louis, MO). Raclopride was dissolved in 0.9% saline, and SCH23390 was dissolved in water. Free-base drug weights were used
in all drug calculations. For PPI testing, injections of 3.0 mg/kg
raclopride or saline were given intraperitoneally, and 1.0 mg/kg
SCH23390 or water was injected subcutaneously immediately before
behavioral testing. For locomotor pattern assessments, injections of
0.1 mg/kg raclopride or saline were given intraperitoneally, and
injections of 0.01 mg/kg SCH23390 or water were given subcutaneously 10 min before behavioral testing. All injections were given at a volume of
5 ml/kg of body weight.
Apparatus. Startle reactivity was measured using four
startle chambers (SR-LAB; San Diego Instruments, San Diego, CA). Each chamber consisted of a clear nonrestrictive Plexiglas cylinder resting
on a platform inside a ventilated box. A high-frequency loudspeaker
inside the chamber produced both a continuous background noise of 65 dB
and the various acoustic stimuli. Vibrations of the Plexiglas cylinder
caused by the whole-body startle response of the animal were transduced
into analog signals by a piezoelectric unit attached to the platform.
These signals were then digitized and stored by a computer. Sixty-five
readings were taken at 1 msec intervals, starting at stimulus onset,
and the average amplitude was used to determine the acoustic startle
response. Sound levels in decibels (A) were
measured as described previously (Dulawa et al., 1997), and the SR-LAB
calibration unit was used routinely to ensure consistent stabilimeter
sensitivities between test chambers and over time (Geyer and Swerdlow,
1998).
The video-tracker (VT) consisted of four adjacent white Plexiglas
enclosures (41 × 41 × 34 cm) surrounded by a plastic
curtain. Each mouse was tested individually in a separate enclosure. A video camera, mounted 158 cm above the enclosures, provided the signal
for the Polytrack digitizer (San Diego Instruments). The signal was
processed to obtain the left-uppermost coordinate for each of the four
animals simultaneously. The signal was stored in a PC computer for
further off-line processing. For this investigation, the position
(x, y) (in pixels) of each animal was sampled at a rate of 18.18 Hz and used to generate a coordinate file
(x, y, t) consisting of the
x-location, the y-location, and the duration of
time (t) spent at that location. The spatiotemporal
resolution of each event recorded was 0.32 cm, 0.32 cm, and 55 msec,
which corresponded to a maximum speed of 25 cm/sec.
Startle and prepulse inhibition session. All PPI test
sessions consisted of startle trials (PULSE-ALONE), prepulse trials (PREPULSE+PULSE), and no-stimulus trials (NOSTIM). The PULSE-ALONE trial consisted of a 40 msec 120 dB pulse of broadband noise. The
PREPULSE+PULSE trials consisted of a 20 msec noise prepulse, a 100 msec
delay, then a 40 msec 120 dB startle pulse (120 msec onset-to-onset
interval). Prepulse intensities were 4, 8, and 16 dB above the 65 dB
background noise. The NOSTIM trial consisted of background noise only.
Each test session began and ended with five presentations of the
PULSE-ALONE trial; in between, each trial type was presented 10 times
in a pseudorandom order. The time between trials averaged 15 sec
(range, 12-30 sec). After the mice were placed in the startle
chambers, a 65 dB background noise level was presented for a 10 min
acclimation period and continued throughout the test session. Each
animal was always tested in the same startle chamber.
After the initial characterization of locomotor activity, mice were
tested in a baseline session to determine PPI and startle reactivity
levels. Mice were then assigned to receive either drug or vehicle
(balanced for startle chamber assignment and treatment) and were tested
in the PPI session. The amount of PPI was calculated as a percentage
score for each prepulse trial type: % PPI = 100 {[(startle response for PREPULSE+PULSE)/(startle response for PULSE-ALONE)] × 100}. Startle magnitude was calculated as the average response to all of the PULSE-ALONE trials, excluding the first
and last blocks of five PULSE-ALONE trials. ANOVAs were used to compare
means; where applicable, Tukey's tests were used for post
hoc analysis. If there were no significant main effects or
interactions with gender, data from males and females were combined for
analysis. The computations were performed using the BMDP
statistical package (SPSS, Inc., Chicago, IL).
For brevity, main effects of prepulse intensity (which were always
significant) will not be discussed. PPI was also analyzed using
difference scores, where PPI Difference = (PULSE-ALONE PREPULSE+ PULSE) for each prepulse trial type. Using these difference scores, the same ANOVAs were performed, and the same effects of genotype and genotype by drug treatment interactions were found (data
not shown). Data from the NOSTIM trials are not included in Results
because the values were negligible relative to values on trials
containing startle stimuli. The habituation of the startle response was
investigated by grouping the startle trials into four blocks (five
PULSE-ALONE trials each, in order of presentation) and measuring the
decrease of startle magnitudes across blocks. ANOVAs revealed
significant main effects of block, indicating normal habituation of the
startle response, but there were no consistent interactions between
block and genotype to suggest that the DAT (/) mice had any
impairment in startle habituation (data not shown).
Locomotor pattern testing. The DAT mice were characterized
initially in the VT enclosure before any PPI testing. Each mouse was
placed in the bottom left-hand corner of an enclosure at the start of
the test session. The movements of the mice were tracked for either 15 or 30 min, with data being stored in three or six 5 min blocks,
respectively. Two categories of measures were obtained. First, the
amount of locomotor activity was measured by the distance traveled,
i.e., tracing the consecutive locations of the animal using the highest
resolution of the video-tracker and calculating the distance between
them. Second, the geometric patterns of locomotor activity were
quantified by the spatial scaling exponent, d, as described
in detail elsewhere (Paulus and Geyer, 1991a). Briefly, the
spatial scaling exponent quantifies the extent to which a sequence of
movements falls along a straight line (d = 1) or within a
circumscribed area (d = 2). The locomotor activity data were analyzed based on a genotype-by-time or a genotype-by-drug-by-time ANOVA.
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RESULTS |
Prepulse inhibition
As hypothesized, the DAT (/) mice exhibited a consistent
deficit in sensorimotor gating, as measured by the PPI of the startle response (Fig. 1). There was a main
effect of genotype [F(2,76) = 8.3;
p < 0.01] on PPI and no interaction with prepulse
intensity [F(4,152) = 0.3; NS].
Post hoc comparisons revealed that the DAT (/) group
differed significantly from both the DAT (+/+) and DAT (+/) groups of
mice at the 4 dB [F(2,76) = 6.2;
p < 0.01], 8 dB
[F(2,76) = 6.2; p < 0.01], and 16 dB [F(2,76) = 6.8;
p < 0.01] prepulse intensities. This deficit in PPI
in the transporter knock-out mice was not associated with any
significant alteration in startle reactivity (Table
1).
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Figure 1.
Baseline PPI levels in DAT mice from the first
cohort. PPI was significantly disrupted in the DAT knock-out (/)
mice at each prepulse intensity, compared with both DAT (+/+) and DAT
(+/) mice (** p < 0.01). Values represent mean
% PPI ± SEM.
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When the three groups of DAT mice were treated with the DA D2
receptor antagonist, raclopride, PPI was increased significantly in the
DAT (/) and (+/) mice, but not in the (+/+) mice (Fig. 2). There were main effects of genotype
[F(2,75) = 10.0; p < 0.01] and drug treatment [F(1,75) = 30.0; p < 0.01], as well as a significant genotype by
treatment interaction [F(2,75) = 4.7;
p < 0.01]. To determine the source of this
interaction, data were segregated by genotype, and two-way ANOVAs were
completed with drug treatment and prepulse intensity being
within-subjects factors. As expected from studies in rats (Swerdlow et
al., 1991), raclopride had no effect in the DAT (+/+) mice (Fig. 2). In
contrast, the DAT (/) mice showed a significant main effect of drug
treatment [F(1,8) = 12.8;
p < 0.01], reflecting a raclopride-induced increase
in PPI (Fig. 2). In addition, the DAT (+/) mice also showed
significant increases in PPI after raclopride treatment
[F(1,45) = 12.6; p < 0.01] (Fig. 2). There were no significant effects of genotype or
raclopride treatment on startle reactivity in the DAT mice (Table
1).
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Figure 2.
PPI levels in DAT mice after pretreatment with 3.0 mg/kg raclopride. Raclopride had no effect on PPI in the DAT (+/+)
mice; however, raclopride significantly increased in both the DAT
(/) and (+/) mice (p < 0.01). Values
represent mean % PPI ± SEM.
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To determine the reliability of these phenotypic differences, a problem
that has been raised as a general concern for all studies with mutant
mice (Crabbe et al., 1999), a second cohort of DAT mice was examined.
In this cohort, equivalent deficiencies in PPI were found in the DAT
(/) mice. The large sample size of this cohort enabled us to
determine that gender significantly affected startle plasticity.
Because there was a significant main effect of gender on PPI
[F(1,95) = 7.1; p < 0.01] and startle reactivity
[F(1,95) = 10.1; p < 0.01], we considered the startle data from the female and male mice
separately. Regardless of these gender differences, both the female and
male DAT (/) mice still displayed decreased levels of PPI. There
was a significant main effect of genotype on PPI in both female
[F(2,56) = 10.0; p < 0.01] and male [F(2,52) = 20.5;
p < 0.01] mice (Fig.
3A,B).
Similar to the previous cohort, further analysis revealed that female DAT (/) mice had significantly lower PPI compared with (+/+) controls at the 4 dB [F(2,56) = 3.5;
p < 0.05], 8 dB
[F(2,56) = 8.5; p < 0.01], and 16 dB [F(2,56) = 13.6;
p < 0.01] prepulse intensities; comparable
disruptions in PPI were also found in the male DAT (/) mice at the
4 dB [F(2,52) = 6.4;
p < 0.01], 8 dB
[F(2,52) = 22.2; p < 0.01], and 16 dB [F(2,52) = 22.5;
p < 0.01] prepulse intensities. Although female DAT
mice had comparable levels of startle reactivity, there was a
significant main effect of genotype on startle reactivity in the male
DAT mice [F(2,52) = 10.9;
p < 0.01], in which startle reactivity tended to be
higher for the DAT (+/) and lower for the DAT (/), compared with
(+/+) controls (p < 0.1) (Table 1).
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Figure 3.
Baseline PPI levels in the second cohort of female
and male DAT mice. Despite a gender effect, PPI was significantly
disrupted in the female (A) and male
(B) DAT knock-out (/) mice at each prepulse
intensity compared with both DAT (+/+) and (+/) mice
(*p < 0.05, ** p < 0.01).
Values represent mean % PPI ± SEM.
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In an attempt to restore PPI and further characterize the role of the
D1 receptor in the mediation of PPI, the male DAT mice were tested with
the DA D1 receptor antagonist, SCH23390. The DAT (/) mice, however,
continued to have lower levels of PPI compared with (+/+) mice,
evidenced by a main effect of genotype on PPI
[F(2,52) = 12.9; p < 0.01] (Fig. 4). There were no main effects of genotype or drug treatment on startle reactivity in the male
DAT mice (Table 1).
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Figure 4.
PPI levels in DAT mice after pretreatment with 1.0 mg/kg SCH23390. SCH23390 had no effect on PPI in the DAT (+/+), (+/),
or (/) mice. Values represent mean % PPI ± SEM.
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Motor behavior analysis
Figure 5 shows a 5 min sample of the
movement patterns from each of the three genotypes during the test
session in the video-tracker enclosure from the first cohort of DAT
mice. The movement patterns of the DAT (+/+) and (/) mice reflect
the profound differences in the sequential organization of their
behavior. The wild-type mouse exhibited a mixture of straight,
meandering, and circumscribed movements around the enclosure. In
contrast, the homozygous mouse engaged almost exclusively in repetitive
straight movements around the perimeter of the enclosure. The
heterozygous mutant exhibited bouts of both repetitive straight
movement and varied movement patterns. These differences were confirmed
quantitatively by the spatial scaling exponent, d, a measure
based on fractal geometry, which quantifies the degree to which
sequences of movements are straight (d = 1) or circumscribed
(d = 2) (Paulus and Geyer, 1991b). The significant effect of
genotype on the d measure
[F(2,74) = 8.1; p < 0.01] confirmed the reliability of the observed pattern differences.
There was a significant time-by-genotype interaction [F(4,148) = 2.5; p < 0.05]; post hoc analysis revealed that the average pattern
did not differ across genotype during the first 5 min of testing. In
contrast, during the second and third 5 min blocks, both the DAT (+/)
and (/) mice exhibited significantly straighter movements (lower
spatial d values) when compared with (+/+) controls
(p < 0.05 and p < 0.01, respectively), with the (+/) being significantly higher than the
(/) (p < 0.05). Thus, the interaction
between genotype and time is attributable to the fact that the DAT
(+/) and (/) mice, relative to DAT (+/+) mice, show an attenuated
transition from predominantly straight movements to predominantly
circumscribed movements that typically accompanies the habituation of
locomotor activity in a novel environment (Paulus et al., 1999).
Confirming previous reports (Giros et al., 1996), a significant main
effect of genotype on locomotor activity was found
[F(2,74) = 26.8; p < 0.01]. DAT (/) mice were more active than the DAT (+/+) and (+/)
mice in the open field (p < 0.01), with the DAT
(+/) levels of locomotor activity falling between the (+/+) and
(/) levels.
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Figure 5.
Baseline characterization of the patterns of
locomotor activity. The DAT (+/+) mouse (left panel) explored all parts
of the video-tracker enclosure and engaged in both circumscribed and
straight movements, particularly near the corners of the enclosure. In
contrast, the DAT (/) mouse (right panel) engaged in repetitive
straight movements along the walls of the enclosure and rarely ventured
into the center region. The DAT (+/) mouse (center panel) showed a
pattern that contained elements of occasional meandering and
circumscribed movements and some repetitive straight movements in the
periphery of the enclosure. Each pattern was reconstructed from a
representative mouse from each genotype using the first 5 min of
data.
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The same perseverative motor phenotype was found in the second cohort
of DAT mice, in which the DAT (/) mice had markedly different
patterns of motor activity as measured by spatial d at
baseline testing. The larger sample size of the second cohort enabled
us to examine whether the sequential organization of behavior differed
across gender. Indeed, male and female mice differed in their patterns
of locomotor behavior [F(1,98) = 6.3;
p < 0.01]. Specifically, female mice, when compared
with males, showed straighter movement sequences in the test
enclosures. In addition, the spatial d values for the
heterozygous (+/) female mice showed an intermediate level of motor
patterning, composed of circumscribed and meandering movements as well
as straight movement sequences. Consequently, the motor activity data
were separated by gender for further analysis. As seen in Figure
6A, the female DAT
(/) mice were more perseverative in their motor behavior than (+/+)
controls, with significant main effects of genotype
[F(2,52) = 18.2; p < 0.01] and time [F(5,260) = 6.3;
p < 0.01] on spatial d. The female DAT
(/) mice moved in straighter sequences (lower spatial d
values) than the DAT (+/+) (p < 0.01) or the
(+/) (p < 0.05) mice in each block of time.
The female DAT (/) mice were also hyperactive, supported by a
significant main effect of genotype on the amount of activity in the
female DAT mice [F(2,49) = 19.7;
p < 0.01] and a time-by-genotype interaction
[F(10,245) = 2.9; p < 0.01]. The female DAT (/) mice were more active compared with
either the (+/+) or (+/) mice in each block (p < 0.01), except for the second block in which there was a trend toward
the (/) mice traveling more than the (+/+) mice (Fig.
6B).
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Figure 6.
Measures of locomotor behavior in the DAT mice.
The spatial scaling exponent, d, is based on the concept
of fractal geometry and describes the roughness or straightness of the
path by relating the number of movements to the distance between these
movements. Specifically, a d value of 1 indicates that
successive movements follow a straight line, whereas a d
value of 2 signifies a highly circumscribed movement pattern.
A, In the female mice, the movement patterns of DAT
(/) mice were characterized by a significantly reduced
d value in each block, indicating a predominance of
repetitive straight movement sequences (**p < 0.01). DAT (+/) mice exhibited locomotor patterns that were
intermediate to the DAT (+/+) and (/) mice in block 1 [*p < 0.05, compared with DAT (+/+) mice;
 p < 0.05, compared with DAT (/)
mice]. C, In the male mice, the DAT (/) mice also
displayed significantly straighter sequences of motor behavior,
indicated by lower spatial d values compared with (+/+)
control mice (**p < 0.01). Both the female
(B) and male (D) DAT
(/) mice were significantly more active than the DAT (+/+) and
(+/) mice across the test session (**p < 0.01).
Values represent means ± SEM for six 5 min blocks.
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Similar findings of perseverative and hyperactive motor behavior were
also found in the male DAT mice. There were significant main effects of
gene [F(2,46) = 36.9;
p < 0.01] and time
[F(5,230) = 3.2; p < 0.01] on spatial d, with interactions between genotype and
time [F(10,230) = 4.0;
p < 0.01]. There were no significant differences
between genotypes in the first 5 min block, but the DAT (/) mice
moved in significantly straighter sequences (lower spatial d
values) than either the (+/+) or (+/) mice (p < 0.01) for the rest of the test session (Fig. 6C). The
male DAT (/) also displayed increases in activity, confirmed by
main effects of genotype [F(2,54) = 38.8; p < 0.01] and time
[F(5,270) = 7.6; p < 0.01], as well as a time-by-genotype interaction
[F(10,270) = 7.7; p < 0.01]. Like the female mice, the male DAT (/) mice were
hyperactive compared with either the DAT (+/+) or (+/) mice in each
block of time (p < 0.01) (Fig.
6D).
To examine whether the changes in locomotor patterns induced by the
removal of the DA transporter were related to an increased occupancy of
the D2 receptor, a DA D2 receptor antagonist was administered before
testing. As seen in Figure 7, raclopride
significantly affected the amount of locomotor activity in the DAT
(/) mice, but these effects were not parallel to those observed for
the patterns of locomotor behavior. In heterozygous (+/) mice,
however, raclopride differentially and time-dependently affected the
locomotor patterns. Specifically, although neither (+/+) nor DAT
(/) mice exhibited changes in locomotor patterns, DAT (+/) showed
a significant increase in spatial d, indicating a more
circumscribed movement pattern (Figs. 7,
8A). There were
significant main effects of genotype
[F(2,43) = 45.5; p < 0.01] and time [F(5,215) = 16.4; p < 0.01] on spatial d, with interactions
between block and genotype [F(10,215) = 6.1; p < 0.01], block and drug treatment
[F(5,215) = 7.8; p < 0.01], and time and genotype and drug treatment
[F(10,215) = 4.0; p < 0.01]. There was no effect of raclopride in either the (+/+) or
(/) mice, but raclopride made the DAT (+/) mice more local in
their motor behavior (increased spatial d) in blocks 1, 2, and 6 (p < 0.01) (Fig. 8A).
As during baseline testing, the vehicle-treated DAT (/) mice moved
in significantly straighter sequences (lower spatial d
values) at blocks 2-6 (p < 0.01), with a trend
toward straighter movements in block 1 (p < 0.10), when compared with (+/+) controls (Fig. 8A).
The vehicle-treated (/) mice were also more active than the (+/+)
controls, and, as expected from previous studies (Paulus and Geyer,
1991a), the D2 receptor antagonist significantly reduced the
hyperactivity in the DAT (/) mice. There were significant main
effects of genotype [F(2,43) = 89.7;
p < 0.01] and time
[F(5,215) = 4.0; p < 0.01] on the amount of motor activity, with block-by-genotype
[F(10,215) = 6.4; p < 0.01], block-by-drug [F(5,215) = 10.7; p < 0.01], and block-by-genotype-by-drug [F(10,215) = 3.8; p < 0.01] interactions, as well as a nearly significant genotype by
drug [F(2,43) = 3.0;
p = 0.06] interaction. Raclopride significantly
reduced motor activity in the DAT (/) mice in block 1 (p < 0.01) and block 2 (p < 0.05), although they were still more
active than the (+/+) controls (p < 0.01) (Fig. 8B). In contrast, raclopride had no effect in either
the (+/+) or (+/) mice.
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Figure 7.
Five min sampling of patterns of locomotor
activity in DAT mice treated with vehicle, SCH23390, or
raclopride.
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Figure 8.
Effects of 0.1 mg/kg raclopride on spatial
d and amount of locomotor activity. A, In
the female mice, the movement patterns of DAT (/) mice were not
affected by pretreatment with the D2 antagonist raclopride.
B, The amount of locomotor activity was significantly
reduced, however, in the DAT (/) mice pretreated with raclopride in
blocks 1 and 2 (*p < 0.05, **p < 0.01), while having no effect in either the DAT (+/+) or (+/)
mice. Values represent means ± SEM for six 5 min blocks.
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In a separate experiment, the effect of reducing DA occupancy at
the D1 receptor was examined by pretreatment with SCH23390. If D1
receptors are critical for the distance-covering movements, which has
been hypothesized for rats previously (Paulus et al., 1993), one would
expect an increase in spatial d regardless of changes in
locomotor activity in mice that exhibit an increased dopaminergic tone.
Specifically, it was hypothesized that DAT (/) and DAT (+/) but
not (+/+) mice should show an increase in spatial d after
the administration of SCH 23390. When the male DAT mice from the second
cohort were challenged with DA D1 receptor antagonist SCH23390, the DAT
(/) mice were less perseverative and less active in their motor
behavior (Fig. 7). Main effects of genotype
[F(2,43) = 27.1; p < 0.01], drug [F(1,43) = 38.0; p < 0.01], and block
[F(5,215) = 94.3; p < 0.01] were found on spatial d, with interactions between
genotype and drug [F(2,43) = 9.2;
p < 0.01], time and drug
[F(10,215) = 16.1; p < 0.01], and time and genotype and drug
[F(10,215) = 3.1; p < 0.01]. The vehicle-treated DAT (/) mice maintained more
straight patterns of motor behavior (lower spatial d values)
in blocks 1-6 compared with both (+/+) and (+/) mice
(p < 0.01) (Fig.
9A). SCH23390 significantly
increased spatial d in the DAT (/) mice in each block
(p < 0.01), inducing more local and meandering
behavior, while having no effect in either the (+/+) or (+/) mice
(Fig. 9A). D1 receptor antagonism also reduced the DAT
(/) hyperactivity, confirmed by significant main effects of
genotype [F(2,43) = 61.7; p < 0.01], drug treatment
[F(1,43) = 47.0; p < 0.01], and time [F(5,215) = 40.8;
p < 0.01] on the amount of activity displayed by the
DAT mice, with genotype-by-drug treatment
[F(2,43) = 21.0; p < 0.01], time-by-drug treatment
[F(5,215) = 14.9; p < 0.01], and time-by-genotype-by-drug
[F(10,215) = 4.9; p < 0.01] interactions. The vehicle-treated DAT (/) mice continued
to show hyperactivity compared with both the vehicle-treated (+/+) and
(+/) mice in each block (p < 0.01), whereas
SCH23390 significantly reduced the amount of activity in the DAT
(/) mice in blocks 2-6 (p < 0.01) (Fig.
9B).
View larger version (24K):
[in this window]
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|
Figure 9.
Effects of 0.01 mg/kg SCH23390 on spatial
d and amount of locomotor activity. A, In
the male mice, the D1 antagonist SCH23390 significantly attenuated the
perseverative motor patterns displayed by the DAT (/) mice across
the test session (**p < 0.01). B,
D1 receptor antagonism also reduced the amount of locomotor activity in
the DAT (/) mice in blocks 2-6 (**p < 0.01).
Values represent means ± SEM for six 5 min blocks.
|
|
| |
DISCUSSION |
In this study, two separate cohorts of DAT (/) mice exhibited
two behavioral phenotypes: a profound disruption of sensorimotor gating
and highly perseverative locomotor patterns. Unlike either DAT (+/+) or
(+/) mice, DAT (/) mice had disrupted PPI, which was reversed
significantly by pretreatment with the preferential D2 receptor
antagonist raclopride. This result supports the hypothesis that D2
receptors are necessary for the dopaminergic modulation of PPI and is
in line with previous findings in both rats and mice (Mansbach et al.,
1988; Peng et al., 1990; Swerdlow et al., 1991; Ralph et al., 1999).
The selective DA D1 receptor antagonist, SCH23390, however, was
ineffective at increasing DAT (/) PPI, suggesting that D1 receptors
are not necessary for the dopaminergic modulation of PPI. The DAT
(/) mice also displayed nonfocal perseverative motor patterns of
behavior, repeatedly moving in straight sequences, compared with DAT
(+/+) control mice. The DAT (/) patterns of motor behavior were
affected differentially by DA receptor antagonists: D2 receptor
blockade had no effect on the perseverative patterns, whereas D1
receptor antagonism augmented the perseverative nature of the motor
behavior. These results demonstrate that D1 receptors are critically
involved in the expression of repetitive perseverative movements in DAT (/) mice. As previously reported (Giros et al., 1996), DAT (/) mice were also found to be hyperactive, and both D1 and D2 receptor blockade significantly reduced the amount of motor behavior produced by
the (/) mice.
Stimulation of the dopaminergic system via blockade of the transporter
or by direct agonists disrupts PPI in rodents (Mansbach et al., 1988;
Dulawa and Geyer, 1996). Accordingly, DAT (/) mice exhibited
profound deficits in PPI, presumably because of their hyperdopaminergic
tone. Of note was the lack of disruption in PPI in DAT (+/) mice,
which have an intermediate expression of the DAT and a corresponding
intermediate elevation of extracellular DA levels [twofold increase
compared with fivefold in DAT (/)], as well as decreases in DA
release, uptake, and tissue concentrations relative to DAT (+/+) and
(/) mice (Jones et al., 1998). Hence, a gene-dosage effect does not
appear to be present in the PPI phenotype in these mice. Instead,
profound changes in the DA system in DAT (/) mice appear to be
necessary to produce a disruption in sensorimotor gating in mice. These
results are consistent with pharmacological studies which have shown
that high doses of DA agonists are necessary to disrupt PPI in mice
(Dulawa and Geyer, 1996; Ralph et al., 1999). Although no gene-dosage
effect was present in the PPI phenotype, it should be noted that others
have reported the presence of gene-dosage effects in other phenotypes not involving behavioral measures (Jones et al., 1999).
Disruptions in PPI produced by DA agonists are reversed by DA D2
receptor antagonists (Mansbach et al., 1988; Swerdlow et al., 1991).
Consistent with these previous reports, the D2 receptor antagonist
raclopride reversed the PPI deficits in the DAT (/) mice without
affecting startle reactivity. Furthermore, the dose of raclopride used
to restore PPI in DAT (/) mice had no effect on PPI in the DAT
(+/+) mice. In preliminary studies, the clinically effective
antipsychotic haloperidol, which has a high affinity for the D2
receptor, also significantly reversed deficient PPI observed in a
separate cohort of DAT (/) mice (Ralph et al., 1997). The
fact that the DAT (/) has 50% downregulation of D2 receptors
(Giros et al., 1996) and loss of autoreceptor function (Jones et al.,
1999) did not appear to affect the ability of raclopride to reverse the
PPI deficit in the mutants, indicating that the D2 receptor system is
still functional despite the profound changes found in the DAT (/)
mice. Although DA D2 receptor antagonism significantly increased PPI in
the DAT (/) mice, the preferential DA D1 receptor antagonist,
SCH23390, had no effect on PPI. Previous studies have suggested that
the DA D1 receptor does not have an independent role in the modulation
of PPI in rats but rather acts together with the DA D2 receptor (Peng
et al., 1990; Wan et al., 1996). Our data suggest that a similar
synergistic mechanism may be present in mice, although further
pharmacological studies are necessary to confirm this conclusion. In
sum, the improvement in sensorimotor gating in the DAT (/) mice by
D2 receptor antagonists corroborates evidence that the PPI-disruptive
effects of amphetamine are absent in D2, but not D3 or D4, receptor
knock-out mice (Ralph et al., 1999) and further supports the specific
role of D2 receptors in the modulation of PPI.
The locomotor activity of DAT (/) mice was characterized by
repetitive, perseverative straight movements in the periphery of the
enclosure. In contrast to both DAT (+/+) and (+/) mice, these animals
did not sample the entire enclosure; rather, the DAT (/) showed a
restricted repertoire of locomotor behavior. This restriction of the
behavioral repertoire is similar to the behavioral patterns that are
observed in rats treated with the direct D1/D2 agonist apomorphine
(Geyer et al., 1986, 1987; Paulus and Geyer, 1991a). The D2 receptor
antagonist raclopride attenuated the locomotor hyperactivity but did
not significantly alter the perseverative movement patterns of DAT
(/) mice. Interestingly, raclopride produced more perseverative
patterns in the DAT (+/) mice compared with vehicle-treated (+/)
mice but not in DAT (+/+) or (/) mice. Thus, blocking the D2
receptor in a mouse with mild hyperdopaminergia, which may result in a
relative increase of D1 occupancy, increased perseverative locomotor
patterns. This finding is consistent with the effect of the D1
antagonist SCH23390 to reduce the perseverative locomotor patterns in
the hyperdopaminergic DAT (/) mouse. Therefore, with increased
dopaminergic tone, occupancy of D1 receptors may be critical in
regulating the perseverative aspect of locomotor behavior in mice.
These findings are also consistent with pharmacological evidence that
antagonism of DA D1 receptors, but not D2 receptors, blocks the
perseverative locomotor behavior induced by amphetamine (Fritts et al.,
1997). Furthermore, the motor patterns in rats treated with the D2/D3
receptor agonist, quinpirole, become straighter when coadministered
with the D1 agonist, SKF38393 (Eilam et al., 1991). Taken together,
these findings provide further evidence that D1 receptor activation is
a critical substrate for the expression of perseverative movement patterns. Although both receptors appear to regulate how frequently behaviors are expressed, the D1 receptor may be critical to how behaviors are organized. Indeed, it has been suggested that
perseverations are linked to prefrontal modulation of ongoing behaviors
(Crider, 1997), a hypothesis that may be related to the important role of D1 receptors in prefrontal cortical function. Therefore, prefrontal D1 receptors may provide a molecular target to adjust the extent or
degree of perseverative behavior.
Although there has been some recent criticism of the reliability of
behavioral phenotypes in genetically altered mice (Crabbe et al.,
1999), we have now observed these two striking behavioral phenotypes in
three separate cohorts of DAT mice (Paulus et al. 1997; Ralph et al.,
1997). Although deficits in sensorimotor gating and
perseverative patterns of motor behavior were seen consistently in both
the female and male DAT (/), some gender differences were found.
When a large cohort of DAT mice was tested, we identified significant
gender differences in both PPI and startle reactivity. Male DAT mice
had higher PPI and startle reactivity than female mice. The female DAT
(/) mice also moved in straighter sequences (lower spatial
d values) compared with male DAT (/) mice. In contrast
to the PPI phenotype, there was a gene-dosage effect on the patterns of
locomotor activity. Specifically, female (+/) mice displayed an
intermediate alteration of movement patterns characterized by bouts of
both repetitive straight movements and meandering or circumscribed
movements. Although further experiments will need to clarify the role
of gender in the gene-dosage effect on patterns of locomotor behavior,
these results underscore the importance of examining the behavioral
phenotype of a mutant mouse in both genders. Although we used a
relatively large number of mice in these experiments, which provided
sufficient power to reliably detect phenotypic differences between DAT
(+/) and (/) female mice, some of the phenotypic differences may
be too small to be detected reliably. Therefore, a differential effect
size may provide an alternative explanation for the gender-dependent gene-dosage effect.
When the DA system is functioning normally, as in the (+/+) mice,
behavioral organization is intact, and normal motor activity and gating
can occur. If, however, the DA system is perturbed, in this case
through the hyperdopaminergic state produced by the DAT mutation, there
appears to be a disruption in the processes that are involved in both
the modulation of incoming stimuli and the appropriate expression of
motor responses. Furthermore, there seems to be a differential DA
receptor modulation of these behavioral phenomena, in which the D2
receptor is a key modulator of deficits in PPI and the D1 receptor
modulates the perseverative motor patterns in the DAT (/) mice.
Several psychiatric populations display both deficits in PPI and
perseverative behaviors, including schizophrenia, schizotypal
personality disorder, obsessive-compulsive disorder, Tourette's
syndrome, and attention deficit hyperactivity disorder (Braff et al.,
1992; Cadenhead et al., 1993; Swerdlow et al., 1993; Castellanos et
al., 1996; Swerdlow, 1998). By revealing aspects of the mechanisms that
control the behavioral deficits seen in the DAT (/) mice, critical
information that is necessary to further understand the
pathophysiologies of these disorders and their therapeutic profiles may
be provided.
| |
FOOTNOTES |
Received Aug. 24, 2000; revised Oct. 13, 2000; accepted Oct. 17, 2000.
This work was supported by the Veterans Affairs VISN 22 Mental
Illness Research, Education, and Clinical Center, National Institute on
Drug Abuse Grants DA02925 and DA11277, and National Institute of Mental
Health Grants F31-MH12806, MH61326, and MH42228. M. A. Geyer holds
an equity interest in San Diego Instruments. M. G. Caron is an
investigator for the Howard Hughes Medical Institute. We thank Beth
Gregersen-Coates, Darlene Giracello, Jason Holt, and Susan Suter for
excellent technical assistance.
Correspondence should be addressed to Dr. Mark A. Geyer, Department of
Psychiatry, University of California San Diego, La Jolla, CA
92093-0804. E-mail: mgeyer{at}ucsd.edu.
| |
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ABSTRACT
INTRODUCTION
