Central Nervous System Diseases Research, Pharmacia & Upjohn, Inc.,
Kalamazoo, Michigan (D.L.F., L.M.N., M.P.S., J.S.A., K.A.S., K.M.M.)
and
Department of Psychiatry, Case Western Reserve University,
Cleveland, Ohio (B.K.Y.)
Our studies examined the role of dopamine D4 receptors in the induction
of behavioral sensitization to amphetamine (Amp) and accompanying
neurochemical and molecular adaptive responses using a highly selective
D4 antagonist, PNU-101387G. Behavioral sensitization to an acute
challenge of Amp (2 mg/kg, s.c.) was observed in rats pretreated with
five daily doses of Amp (2 mg/kg/d, s.c.) followed by 7-day withdrawal.
Interestingly, coadministration of PNU-101387G with Amp during
pretreatment completely blocked the sensitized response to an acute Amp
challenge. The behavioral sensitization and its blockade by the D4
antagonist were observed in the absence of significant differences in
cerebellar Amp levels among the various pretreatment groups.
Accompanying behavioral sensitization were two postsynaptic
neuroadaptive responses: reduction in the ability of Amp to induce
c-fos gene expression in the infralimbic/ventral prelimbic
cortex and NT/N mRNA in the accumbal shell. However, concurrent
blockade of D4 receptors during Amp pretreatment prevented the
refractoriness in c-fos and NT/N responsiveness to acute
Amp. We observed also a presynaptic neuroplastic response associated with the behavioral sensitization: a significant augmentation in the
ability of Amp to increase extracellular dopamine concentrations in the
nucleus accumbens shell. As with the behavioral sensitization and
associated postsynaptic adaptive responses, concurrent administration of PNU-101387G with Amp during pretreatment blocked the augmentation in
Amp-induced dopamine release. Taken together, these data demonstrate that dopamine D4 receptors play an important role in the induction of
behavioral sensitization to Amp and accompanying adaptations in pre-
and postsynaptic neural systems associated with the mesolimbocortical dopamine projections.
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Introduction |
Repeated
administration of psychostimulants, like d-Amp, to rodents
produces a progressive and long-lasting increase in behaviors such as
locomotor activity and stereotypy, a phenomenon generally known as
"behavioral sensitization" or "reverse tolerance" (Segal, 1975
,
Post and Contel, 1983). The enduring hypersensitivity to psychostimulants is observed also in humans and is thought to underlie
drug addiction as well as stimulant psychosis (reviewed by Robinson and
Berridge, 1993; Lieberman et al., 1997). Hence, recent
research has focused on identification and characterization of
neuroadaptive responses leading to behavioral sensitization to
stimulants.
The mesolimbic dopamine system plays a critical role in the development
of sensitization to Amp (reviewed by Kalivas and Stewart, 1991; White
and Wolf, 1991; Robinson, 1991) which acts primarily by releasing
dopamine. Studies of microinjections of the stimulant in discrete brain
regions indicate that the initiation of sensitization may be produced
by the dopamine-releasing effect of Amp in the origin of the mesolimbic
dopamine neurons, the VTA (Kalivas and Weber, 1988; Vezina, 1993; Cador
et al., 1995). Activation of D1 (D1, D5) but not D2 (D2, D3,
D4) receptors in the VTA is proposed to initiate sensitization since
administration of a D1-like antagonist (but not D2-like antagonist)
directly into the VTA blocks the development of sensitization to
peripherally administered Amp (Stewart and Vezina, 1989; Bjijou
et al., 1996). However, the involvement of D2-like receptors
in the initiation of methamphetamine-induced behavioral sensitization
is evident in a number of studies in mice using peripheral
administration of nonselective D2 antagonists (Ujike et al.,
1989; Kuribara, 1994; Kuribara, 1996). It is possible that in addition
to the VTA D1 receptors, D2-like receptors expressed at a site(s)
outside the VTA, participate in the induction of sensitization to Amp.
This is supported by a recent report by Karler et al. (1997)
who demonstrated that direct administration of D2-like antagonists,
sulpiride or spiperone, into the anterior frontal cortex blocked the
induction of sensitization to Amp in mice.
The D2-like receptors are encoded by three distinct genes termed, D2,
D3 and D4 (Bunzow et al., 1988; Sokoloff et al.,
1990; Van Tol et al., 1991). The studies cited above
examined the role of D2 receptors in induction of sensitization using
antagonists that did not distinguish among members of the D2 receptor
family. Hence, the role of each subtype of D2-like receptors in
stimulant sensitization remains unknown. Recently, a D4 receptor
selective antagonist, PNU-101387G (previously termed U-101387G) has
become available and shows pharmacological properties distinct from
those of nonselective D2-like antagonists (Merchant et al.,
1996b). We used PNU-101387G as a tool to understand the role of D4
receptors in the induction of behavioral sensitization to Amp.
The development of behavioral sensitization to Amp is thought to
reflect neuroadaptive biochemical and genomic responses in both pre-
and postsynaptic systems triggered by the first exposure to the
psychostimulant. Presynaptically, one of the more consistent adaptive
responses appears to be an augmentation in the capacity of Amp to
release dopamine in the dorsal neostriatum and the nucleus accumbens
(reviewed in Kalivas and Stewart, 1991). Postsynaptic neuroplasticity
accompanying behavioral sensitization to Amp is evident by alterations
in the expression of transcription factors (e.g.,
c-fos and Fos-like antigens, CREB, NGFI-A), and genes
encoding neuropeptides (e.g., dynorphin, enkephalin,
substance P) (Graybiel et al., 1990; Konradi et
al., 1994; Wang and McGinty et al., 1995a; Jaber
et al., 1995). Hence, our studies examined the effects of an
Amp challenge on 1) c-fos mRNA expression in the medial
prefrontal cortex, 2) expression of the gene encoding the putative
"endogenous neuroleptic," neurotensin, in the nucleus
accumbens-shell and 3) extracellular dopamine levels in both, the shell
and core sectors of nucleus accumbens in rats pretreated with Amp
(i.e., behaviorally sensitized rats). To establish the role
of D4 dopamine receptors in the induction of behavioral sensitization
to Amp and accompanying neuroadaptive responses, we examined the
effects of administration of the D4 selective antagonist, PNU-101387G,
with Amp during the pretreatment phase on Amp challenge-induced
behavioral, neurochemical and genomic responses.
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Materials and Methods |
Animal treatment.
For the behavioral studies, adult, male,
Sprague-Dawley rats (200 g at the beginning of the study) from Charles
River Laboratory (Kalamazoo, MI) were maintained in a controlled
environment with 12-hr light/dark cycle (lights on at 6:30
A.M.) and free access to laboratory food and tap water.
Rats were housed three/cage and after at least 5 days of acclimation
they were divided into the following four pretreatment conditions: 1)
vehicle (2.5% methylcellulose, 1 ml/kg, i.p.) plus vehicle (0.9%
saline, 1 ml/kg, s.c.), 2) PNU-101387G (21 µmol/kg or 10 mg/kg; i.p.)
plus vehicle (saline, 1 ml/kg, s.c.), 3) vehicle (2.5% methyl
cellulose, 1 ml/kg, i.p.) plus d-amphetamine sulfate (5.4 µmol/kg or 2 mg/kg as the salt form, s.c.) or 4) a combination of
PNU-101387G and Amp for 5 days; each i.p. administration was followed
immediately by the respective s.c. injection. The drugs were
administered in the home cages once daily between 8:00 and 9:00
A.M. at the indicated doses. After pretreatment, rats were
withdrawn for 7 days during which they were handled and given a
sham-injection (i.e., needle poke) each day to habituate
them to handling and injection stress. On day 13, rats were habituated
to activity chambers (see below) for 30 min, removed briefly for an
acute challenge injection of 1) vehicle (2.5% methyl cellulose, 1 ml/kg, i.p.) plus vehicle (0.9% saline, 1 ml/kg, s.c.), 2) vehicle
(2.5% methyl cellulose, 1 ml/kg, i.p.) plus Amp (2 mg/kg, s.c.) or 3)
PNU-101387G (10 mg/kg, s.c.) plus vehicle (0.9% saline, 1 ml/kg,
s.c.), placed back in the activity chambers and monitored for 1 hr.
Table 1 shows the pretreatment and
challenge treatment groups used in the study. At the end of the
behavioral recording, rats were killed, trunk blood collected, brains
rapidly removed and frozen on dry ice. Brains were used for in
situ hybridization histochemistry for c-fos and NT/N
mRNA detection as described below. Amp concentrations were determined
in the plasma (isolated from the trunk blood) and cerebellum from each
animal.
For the in vivo microdialysis studies, adult, male
Sprague-Dawley rats (Zivic-Miller) were used. Three groups of rats were used for monitoring extracellular dopamine concentrations: 1) V/A, 2)
A/A, 3) U+A/A. Pretreatment and withdrawal were carried out exactly as
described above for the behavioral studies. The doses of the drugs,
their vehicles and the number of injections received by each animal
were identical to the protocol detailed above.
Measurement of locomotor activity.
Locomotor activity was
monitored using Digiscan Animal Activity Monitoring System running
DigiPro Windows software (Accuscan Instruments, Columbus, OH). The
apparatus consisted of eight transparent Plexiglas activity monitoring
cages (40.5 × 40.5 cm). For the measurement of horizontal
activity, each cage was equipped with 2 sets of 16 photocell arrays
located at right angles to each other, projecting horizontal infrared
beams 2.5 cm apart and 3.75 cm above the cage floor. Another set of 16 horizontal beams placed 14 cm above the cage floor recorded vertical
activity. Each beam break (regardless of the nature of activity) in the
lower array was recorded as a horizontal activity count. Similarly,
beam breaks in the upper array recorded vertical activity counts. The
DigiPro Windows software uses a mathematical algorithm to compute total distance traveled (in cm) and takes into account factors such as the
distance between interrupted beams, the length of the animal in the
horizontal plane and the direction of ambulatory activity (along the
walls vs. diagonal). To help isolate test animals from environmental effects, each cage was placed inside a sound attenuation chamber equipped with a dim light and a fan for a constant background noise. All rats were tested between 7:30 A.M. and 3:30
P.M. Eight activity chambers were used simultaneously with
allocation of rats (from each treatment group) to the cages in a Latin
square design. On the challenge day, rats were placed individually in the LMA chambers and allowed to habituate for 30 min. Animals were then
removed briefly from the activity chambers to receive an acute
challenge of an appropriate substance. After dosing, animals were
replaced in the respective chambers and their activity was monitored
for an additional 60 min. Cumulative counts for horizontal activity,
vertical activity and total distance were computed for the 60-min
period after the challenges. After the behavioral recordings, animals
were killed, brains removed and frozen for in situ
hybridization histochemistry.
In situ hybridization histochemistry.
One half (left or
right, randomly chosen) of each brain was cut into 20-µm sections and
thaw-mounted onto gelatin-coated slides. These were stored at -80°C
until used. Sections were processed as detailed previously (Merchant
et al., 1992). Briefly, slides were air dried, fixed in 4%
paraformaldehyde, rinsed in PBS, acetylated in triethanolamine buffer
containing acetic anhydride, dehydrated through a graded alcohol
series, delipidated with chloroform, dehydrated in 100% ethanol,
hydrated with 95% ethanol and air dried. A c-fos antisense
deoxyoligonucleotide probe complementary to the rat c-fos
mRNA was end-labeled using terminal deoxynucleotidyl transferase and
[35S]-labeled dATP to a specific activity of
7.5 to 15 × 106 dpm/pmol probe. For NT/N
mRNA hybridization, a cRNA probe was transcribed in vitro
and labeled with [35S]UTP to a specific
activity of 40 to 80 × 106 dpm/pmol.
Hybridization was carried out overnight at 37°C for the
c-fos deoxyoligonucleotide probe or 48°C for the NT/N
riboprobe at a concentration of 2 pmol probe/ml of hybridization buffer as detailed before (Merchant et al., 1992). After the
incubation, high stringency washes were conducted in 1X SSC at 65°C
for c-fos and 0.1X SSC at 55°C for NT/N mRNA
hybridization. Slides were dehydrated through a graded alcohol series,
air-dried and apposed to KODAK Biomax-MR film for autoradiography.
After film developing, slides were coated with liquefied NTB-2 emulsion
(Kodak), and developed in Kodak D-19 developer after an appropriate
exposure time. Sections were counterstained with Harris Hematoxylin
from MasterTech (Lodi, CA).
Analysis of hybridization signal was carried out using dark and
bright-field optics at 40x (for the number of labeled cells) or 100x
(for the number of grains/cell) magnification. The number of labeled
cells and the average density of autoradiographic grains per cell was
determined as detailed before (Merchant et al., 1996a). Three atlas-matched (Paxinos and Watson, 1986) sections were used for
each region from each animal. The infralimbic/ventral prelimbic region
(Bregma 2.7 mm) and the shell sector of the nucleus accumbens (Bregma
1.6 mm) (as shown in schematics in figs. 4 and 6) were analyzed for
counting the total number of c-fos or NT/N mRNA expressing cells. The average grain density per cell was determined by analyzing 70 to 100% of the labeled cells in each region. Data from the three
sections were averaged to obtain the value for each animal. Group
averages were computed for statistical analysis of the hybridization signal.
Plasma amphetamine detection.
Trunk blood was centrifuged
(1000 × g for 20 min) to separate plasma and frozen at
-20°C for later analysis of Amp content using HPLC and fluorescence
detection. The assay of Amp was based on the primary amine character of
the chemical. Primary amines can be measured sensitively because they
form fluorescent adducts with o-phthalaldehyde and a thiol.
To 1 ml of plasma 100 µ l of 10 N NaOH and 5 µg of internal
standard were added. The solution was mixed with 2 ml of acetonitrile
and centrifuged. The organic layer was removed and dried using a stream
of nitrogen until 100 µl of solution remained. The concentrated
solution was filtered through a quaternary amine column, and to the
effluent was added 100 µl of the OPA/tBSH reaction mixture (5 mg
of o-phthalaldehyde, 2 µl of t-butylthiol, 100 µl
methanol and 898 µl of .4N Na2Borate). A
100-µl sample was then injected onto an HPLC column. Mobile phase
90% methanol, 10% H2O and 1 g/ml t-butyl
ammonium perchlorate was pumped through a 25 cm ODS column at a rate of
1.5 ml/min. A fluorescence detector was used where EX was 350 and EM
was 420 µm. Calculations were based on the results of a standard
curve normalized to the internal standard.
Cerebellar amphetamine detection.
Frozen brains were weighed
in a 1.5 ml Eppendorf tube and 500 µl of acetone containing 10 µg/ml of internal standard was added. The internal standard was a
phenylcyclohexyl amine analogue of Amp. The mixture was pulse sonicated
to homogeneity and centrifuged. The supernatant was transferred to
another Eppendorf tube, which was placed in a SpeedVac concentrator and
concentrated to about 100 µl. The concentrated solution was filtered
through a quaternary amine column and to the effluent was added 100 µl of the OPA/tBSH reaction mixture. HPLC was conducted as for the
plasma level detection. Data are expressed as milligrams of
d-amphetamine per gram of tissue (mg/g).
Measurement of extracellular dopamine concentrations by in
vivo microdialysis.
Dr. Yamamoto carried out these studies
at Case Western Reserve University. Rats were pretreated with Amp and
withdrawn as described above. On day 4 of the 7-day withdrawal period,
all rats were anesthetized with a combination of xylazine (6 mg/kg, i.m.) and ketamine (70 mg/kg, i.m.). Rats were placed in a stereotaxic apparatus, the skull was exposed and a 1-mm hole was drilled into the
skull above the nucleus accumbens shell (1.8 mm anterior to Bregma, 0.7 mm lateral to the mid-line suture) or core (1.7 mm anterior to Bregma
and 1.6 mm lateral to the mid-line suture) (Paxinos and Watson, 1986).
Dura was carefully removed and a 21-gauge stainless steel guide cannula
was placed stereotaxically into the hole up to the surface of the
cortex without penetrating the brain. The guide cannula with a stylet
obturator was secured to the skull with cranioplastic cement and three
set screws. The tip of the microdialysis probe was located 8.2 or 7.2 mm from the cortical surface for the accumbal shell or core,
respectively.
A concentric-shaped dialysis probe was constructed as previously
described (Yamamoto and Davy, 1992; Yamamoto and Pehek, 1990). The
exposed portion of the dialysis membrane (MW cutoff = 13,000) extended 1.5 mm beyond the tip of the 26- gauge stainless steel tubing.
The microdialysis perfusion flow rate was 2.0 µl/min. A stainless
steel spring tether connected the animal to a liquid swivel. the
perfusion medium was a modified Dulbecco's phosphate-buffered saline
containing 138 nM NaCl, 2.7 nM KCl, 0.5 mM MgCl2,
1.5 mM KH2PO4, 8.1 mM
Na2HPO4, 1.2 mM
CaCl2 and 0.5 mM d-glucose, pH 7.4.
On day 13 after commencing the pretreatment, dialysis probes were
inserted and rats were placed in circular cages that allowed free
movement. After a 4-hr stabilization period, 3 base-line samples
(30-min fractions) were collected and assayed for dopamine by HPLC with
electrochemical detection (Stephans and Yamamoto, 1995). Rats were then
administered an acute challenge of d-amphetamine sulfate (2 mg/kg, s.c.) and the perfusate was collected every 30 min for a total
of 150 min. At the end of 150 min, rats were killed by decapitation,
brains removed and the placement of the probe was determined by
histological examination of the tissue slices. Only those rats with
probe placement into the nucleus accumbens shell or core were included
in the analysis.
Statistical analysis.
There were six to nine animals in each
treatment group. A multifactorial analysis of variance was performed
for each measure (except for locomotor activity where the
time-dependent effects were analyzed by repeated measure analysis of
variance). After a significant difference (P < .05), a multiple
comparisons post hoc test (Fisher's PLSD or Tukey) was
applied to identify groups differing significantly from each other.
| |
Results |
Amphetamine levels in the plasma and cerebellar tissue.
Table 2 contains the plasma levels of Amp
1 hr after an acute Amp challenge to rats pretreated with vehicle, Amp,
PNU-101387G (U) or PNU-101387G plus Amp (U+A). Amp levels were not
measurable in groups that did not receive an acute Amp challenge (data
not shown). The A/A group had significantly higher Amp levels in the plasma than the V/A group. The groups pretreated with PNU-101387G alone
or in combination with Amp showed significantly lower plasma Amp levels
after Amp challenge (U/A or U+A/A) than A/A or V/A groups. Cerebellar
Amp levels were determined to investigate whether the increased plasma
levels of Amp in the Amppretreated group also reflects increased
brain levels of the drug. As shown in table 2, cerebellar Amp levels
did not increase in Amp-pretreated rats compared to the
vehicle-pretreated group (i.e., A/A = V/A). Similarly,
pretreatment with PNU-101387G in combination with Amp had no effect on
cerebellar Amp levels after the psychostimulant challenge.
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TABLE 2
Effects of various pretreatment treatments on plasma/brain Amp
concentrations 1 hr after an acute Amp challenge
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Locomotor activity.
A repeated measure analysis of variance
followed by Fisher's PLSD test showed a significant treatment x time
interaction for horizontal and vertical activity measures [F(55, 325) = 2.11, P < .0001; F(55, 325) = 2.54, P < .0001] as well
as total distance counts [F(55, 325) = 2.13, P < .0001]. Acute
Amp induced motor stimulation in vehicle-pretreated animals (V/A)
primarily as an increase in vertical activity. However, in
Amp-pretreated rats, an Amp challenge (A/A) produced greater increases
in locomotor activity (behavioral sensitization) with a significant
effect in horizontal activity and total distance counts (fig.
1). Challenges with either vehicle or
PNU-101387G produced similar levels of horizontal and vertical activity
in all groups regardless of pretreatment condition. However,
pretreatment with PNU-101387G alone led to a sensitized response to Amp
challenge (U/A) similar to that seen in Amp-pretreated rats. Despite
this, coadministration of PNU-101387G with Amp during pretreatment
blocked the behavioral sensitization (in both, the horizontal activity
counts and total distance traveled) to Amp challenge (i.e.,
U+A/A group). Factorial analysis of variance with cumulative counts
over the one hour period also showed the same pattern of results (fig.
2).
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Fig. 1.
Time course of locomotor activity. Rats were
pretreated with vehicle (V), Amp (A), PNU-101387G (U) or PNU-101387G
plus Amp (U+A) as described in "Materials and Methods." On the
challenge day, rats were habituated to the locomotor activity boxes,
challenged with vehicle, Amp or PNU-101387G and monitored for 60 min
for horizontal activity counts (A), vertical activity counts (B) or
total distance traveled (in cm; C). For simplicity, data from five most
relevant groups are shown. Each point represents cumulative activity
counts for a 10-min period. Statistical analysis was carried out using
repeated measure analysis of variance followed by Fisher's PLSD to
identify groups differing from each other. Note that behavioral
sensitization was observed primarily in horizontal activity counts and
was blocked in animals cotreated with PNU-101387G plus Amp during
pretreatment. **P < .005 and ***P < .0001 vs. V/V, ##P < .001 and ###P < .0001 vs. V/A,  P < .02 and P < .001 vs. A/A.
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Fig. 2.
Cumulative locomotor activity. Behavioral data from
all 12 groups of rats used in our study are shown. The data represent
cumulative horizontal activity (A), vertical activity (B) or total
distance counts (C) during the 60 min of behavioral testing after an
acute challenge. The figure legend represents the four pretreatment
treatments and the horizontal axis denotes the substances administered
during the acute challenge. Each bar represents the group mean ± S.E.M. Behavioral sensitization can be seen as an increase in
horizontal activity counts and total distance traveled (in cm) in
Amp-pretreated rats challenged with Amp as compared to the acute Amp
effects in vehicle-pretreated rats. Note that rats pretreated with
PNU-101387G and Amp showed significantly lower horizontal and vertical
activity counts in response to the acute Amp challenge. Although total
distance counts produced by an acute Amp challenge were reduced in rats
co-treated with PNU-101387G plus Amp, they remained significantly
higher than those in the vehicle/vehicle group did. Statistical
analysis was carried out using multi-factorial ANOVA followed by
Fisher's PLSD test. *P < .05, **P < .01 and ***P < .0001 vs. V/V; ##P < .01 and ###P < .0001 vs. V/A; P < .05, P < .01, P < .0001 vs. A/A.
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Fig. 3.
C-fos mRNA expression in the
infralimbic/ventral prelimbic (IL/vPL) Cortex. Each panel in A is a
representative dark-field, low magnification, photomicrograph through
the IL/vPL cortex (shown schematically in fig. 4) from the four most
critical treatment groups. The groups shown are vehicle/vehicle (V/V),
vehicle/Amp (V/A), Amp/Amp (A/A) and PNU-101387G + Amp/Amp (U+A/A). The
forceps minor of the corpus collosum (c) is labeled to help with
orientation; top of the photomicrographs represents the dorsal side of
the brain. Thus the micrographs from the V/A, A/A and A+U/A groups are
from the left hemisphere and V/V micrograph is from the right
hemisphere. The cellular distribution of autoradiographic grains on
neurons in layer VI is shown in high magnification photomicrographs in
B. Note that acute Amp increased c-fos mRNA expression
in vehicle-pretreated rats and this effect was significantly reduced in
Amp-pretreated animals. Coadministration of PNU-101387G with Amp during
pretreatment prevented the reduced capacity of Amp to induce
c-fos gene expression.
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c-fos mRNA in the IL/vPL cortex.
Acute
administration of PNU-101387G induced c-fos mRNA levels in
the IL/vPL cortex in all groups of rats, regardless of the various
pretreatment conditions. The effect was
evident primarily in deep (V + VI) cortical layers. An acute Amp
challenge also induced c-fos gene expression; the effect was
seen in all layers of the IL/vPL cortex of vehicle-pretreated rats
(V/A). However, a reduction in this response was observed in rats
pretreated with Amp (A/A), i.e., a refractoriness to
Amp-induced c-fos expression accompanied behavioral
sensitization. Pretreatment with PNU-101387G alone did not modulate the
Amp challenge-induced (i.e., U/A group) increases in
c-fos mRNA levels in deep layers but significantly attenuated the Amp response in superficial layers of the IL/vPL cortex.
As with the blockade of behavioral sensitization, coadministration of
PNU-101387G with Amp during pretreatment blocked the refractoriness in
c-fos mRNA response to Amp-challenge (U+A/A) in deep layers of the IL/vPL cortex; the superficial layers showed a partial restoration of Amp-induced c-fos gene induction (figs. 3 and
4).
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Fig. 4.
Quantification of c-fos mRNA
hybridization signal in IL/vPL cortex. The total number of
hybridization-positive cells (A, C) in the IL/vPL cortex and the
average density of autoradiographic grains (B, D) associated with these
cells were quantified as detailed in "Materials and Methods."
Expression of c-fos mRNA in the superficial (layers II
and III, C, D) and deep layers (layers V and VI, A, B) of the IL/vPL
(shaded area in the schematic drawing) is shown. The figure legend
represents the pretreatment treatment and the horizontal axis denotes
the acute challenges. Each bar represents the group mean ± S.E.M.
Note that acute Amp induced c-fos gene expression in
vehicle- but not Amp-pretreated rats. PNU-101387G coadministered with
Amp during pretreatment reinstated the response in deep layers but
failed to do so in the superficial layers of the IL/vPL cortex.
Additionally, pretreatment with PNU-101387G alone blocked the response
to acute Amp in superficial layers of the IL/vPL cortex. *P < .05, **P < .001 and ***P < .0001 vs. V/V;
#P < .05, ##P < .01 and ###P < .001 vs. V/A; P < .05, P < .001 vs. A/A.
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NT/N mRNA levels in the NA-s.
Unlike c-fos gene
expression in the medial prefrontal cortex, an acute challenge of
PNU-101387G produced no changes in NT/N mRNA expression in the NA-s,
regardless of the pretreatment condition. As can be seen in figures
5 and 6,
there was a significant elevation in NT/N mRNA expression in the NA-s
after an acute Amp challenge in vehicle-pretreated rats (V/A). The NT/N
induction in the NA-s by Amp was totally abolished when the Amp
challenge was given to Amp-pretreated rats (A/A). However, animals that
received PNU-101387G plus Amp (U+A/A) during the pretreatment period
showed Amp challenge-induced NT/N mRNA induction that was
quantitatively similar to that observed in vehicle-pretreated rats
(V/A). In contrast to the effects of PNU-101387G pretreatment on
Amp-induced behavior or c-fos mRNA levels in superficial
IL/vPL cortex, basal or Amp-induced NT/N mRNA levels were not altered
in PNU-101387G-pretreated rats.
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Fig. 5.
Neurotensin/neuromedin N (NT/N) mRNA expression in
the NA shell. Film autoradiograms of hybridization to NT/N mRNA are
shown from the following groups: vehicle/vehicle (V/V), vehicle/Amp
(V/A), Amp/Amp (A/A) and PNU-101387G + Amp/Amp (U+A/A). Note that acute
Amp-induced NT/N gene expression was significantly reduced in rats
preexposed to Amp and this effect was blocked by concurrent treatment
of PNU-101387G with Amp during the preexposure period. Arrows indicate
the hybridization signal in the shell sector of the NA.
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Fig. 6.
Quantitation of NT/N mRNA expression in the nucleus
accumbens-shell. The total number of hybridization-positive cells (A)
in the accumbal shell and the average density of autoradiographic
grains (B) associated with these cells were quantified as detailed in
"Materials and Methods." The region of the NA analyzed is shown as
shaded area in the schematic. The figure legend shows pretreatment
treatments and the horizontal axis denotes the acute challenges. Each
bar represents the group mean ± S.E.M. Note an increase in acute
Amp-induced NT/N mRNA levels in vehicle and PNU-101387G + Amp
pretreated rats but not in rats pretreated with Amp alone. *P < .05, **P < .001 and ***P < .0001 vs. V/V;
#P < .05 and ##P < .001 vs. V/A; P < .001 vs. A/A.
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Extracellular dopamine levels in the NA-s and NA-c.
Figure
7 schematically shows the placement of
the dialysis probe in the shell and core sectors of the nucleus
accumbens. Base-line and Amp-induced alterations in extracellular
dopamine levels in the NA-s and NA-c of freely moving rats are shown in
figure 8. As expected, acute
administration of Amp significantly increased extracellular dopamine
concentration in both the shell and the core of the nucleus accumbens
in vehicle-pretreated rats (V/A). In rats preexposed to Amp, there was
an augmentation in Amp challenge-induced extracellular dopamine levels
(i.e.,. A/A group) in the NA-s but not in the NA-c. However,
as with the behavioral sensitization and postsynaptic responsiveness to
acute Amp challenge, coadministration of PNU-101387G with Amp
pretreatment blocked the neural adaptations leading to the augmentation
in dopamine releasing capacity of Amp (U+A/A).
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Fig. 7.
Location of the microdialysis probe placements in
the NA shell and core depicted on a coronal section derived from
Paxinos and Watson (1986). Each line shows the approximate length of
the dialysis membrane. Note that placements in the shell were
restricted to the medial portion of the NA.
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Fig. 8.
Extracellular dopamine concentrations in the core
and shell sectors of the nucleus accumbens. Animals pretreated with
vehicle, Amp or PNU-101387G + Amp were used for the study. After a
stable base line of extracellular dopamine levels, all the groups were
challenged with Amp and 30-min dialysis fractions were collected over a
period of 150 min. Each point represents mean dopamine levels ± S.E.M. Statistical analysis was carried out by repeated measure
analysis of variance followed by Tukey's test for each time point
after the Amp challenge. As shown in the top panel, after the Amp
challenge, all three groups showed comparable increases in
extracellular dopamine concentrations in the accumbal core. However, in
the shell sector (bottom panel), Amp-pretreated groups (A/A) showed
greater augmentation in extracellular dopamine levels than
vehicle-pretreated rats (V/A) and this effect was blocked in rats
cotreated with PNU-101387G + Amp (U+A/A) during the pretreatment phase.
#P < .02 vs. V/A; P < .002 vs. A/A.
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Discussion |
The results demonstrate that dopamine D4 receptors play a critical
role in the induction of behavioral sensitization to Amp in the rat.
The augmentation in locomotor stimulatory effects of Amp produced by
Amp preexposure (pretreatment to Amp followed by a withdrawal period)
was blocked in rats coadministered the D4-selective antagonist,
PNU-101387G, with Amp during pretreatment. In addition, the D4
antagonist blocked pre- and postsynaptic biochemical/cellular alterations accompanying the behavioral sensitization: 1) an
augmentation in the ability of Amp to increase extracellular dopamine
levels in the NA shell, 2) a decrease in the capacity of Amp to induce c-fos mRNA expression in the medial prefrontal cortex and 3)
an attenuation in Amp-induced NT/N mRNA expression in the accumbal shell. Because the effects of PNU-101387G on biochemical, behavioral and cellular alterations accompanying behavioral sensitization to
Amp were observed in the absence of a significant effect on brain
(cerebellar) Amp levels, the data strongly implicate a role of dopamine
D4 receptors in the initiation of Amp sensitization.
Behavioral effects.
The behavioral sensitization to Amp
observed in our study is likely a context-independent phenomenon
because the pretreatment treatments were carried out in the home cages
and the rats were exposed to the locomotor activity monitoring chambers
for the first time on the challenge day. The potential confounding
effects of stress on behavior and c-fos mRNA induction were
minimized by 1) habituating the animals to handling and injections
during the 7-day withdrawal period and 2) habituating them to locomotor activity boxes before administration of acute challenges. The automated
horizontal and vertical activity counts measured in these studies
represent two competing behavioral measures, locomotor activation and
rearing, respectively. To evaluate alterations in focused stereotypies
using the automated system, we examined changes in total distance
traveled as an indirect measure. Total distance in the Digiscan system
computes horizontal ambulatory movement based on horizontal activity
counts (see "Materials and Methods") such that any disproportionate
decrease in total distance (relative to horizontal activity counts)
could indicate a shift to focused stereotypies or rearing behavior.
Thus automated measures of horizontal activity, vertical activity and
total distance counts together offer a comprehensive picture of
qualitative and quantitative changes in exploratory behaviors.
The acute stimulatory effect of Amp (V/A group) was seen primarily in
the form of increased rearing (vertical activity counts). In several
studies since this one, we have observed acute Amp-induced stimulation
as an increase in either horizontal or vertical counts or both and
likely represents heterogeneity among rats and/or an effect of the
relatively short monitoring period (1 hr) necessary for examination of
c-fos gene expression. It is possible that because of the
greater vertical activity response to acute Amp, the sensitization to
Amp was quantitatively and statistically more robust for horizontal
activity and total distance measures (A/A compared to V/A). In rats
that received PNU-101387G with Amp during pretreatment, the behavioral
sensitization in horizontal counts or total distance was not evident.
The effect of PNU-101387G was not simply to shift the expression of
behavior from horizontal activity (i.e., locomotion) to
rearing since there was also a suppression in vertical activity counts
in the U+A/A group when compared to A/A group. The total distance
counts also were lower in the U+A/A group than the A/A group; however,
the degree to which total distance was blocked in rats in the U+A/A
group was no greater than the blockade of horizontal activity counts
(unlike horizontal activity, total distance counts in U+A/A animals
remained significantly greater than those observed in the V/V group
(P < .05) and somewhat higher than the V/A group (P < .15).
Although not definitive, these data argue against the possibility that PNU-101387G blocked the induction of behavioral sensitization (in
horizontal activity) simply by a qualitative shift in behavior to
focused stereotypy.
Interestingly, pretreatment with the D4 antagonist by itself induced
cross-sensitization to Amp in all three behavioral measures. Such a
cross-sensitization to the effects of methamphetamine has been reported
previously in mice pretreated with nonselective D1- or D2-like
antagonists (Kuribara, 1994, 1996; Vezina and Stewart, 1989). The
mechanism underlying the cross-sensitization between PNU-101387G and
Amp is unclear. In recent reports we have demonstrated that the
cross-sensitization is a dose-dependent phenomenon for both PNU-101387G
(K.M. Merchant, Z.-H. Meng and D.L. Feldpausch, unpublished
observations) and nonselective D2 class antagonists, haloperidol,
clozapine (Meng et al., 1997). The lack of sensitization to
Amp in rats pretreated with PNU-101387G plus Amp demonstrates that the
two drugs antagonize the pretreatment effects of each other.
It could be argued that the behavioral sensitization and its modulation
by PNU-101387G involved pharmacokinetic factors since the acute Amp
challenge produced significantly greater plasma Amp levels in A/A rats
compared to the V/A group and this effect was blocked by PNU-101387G
given with Amp during pretreatment (i.e., U+A/A group).
However, several observations argue against this possibility. First,
concurrent with an increase in plasma Amp level, there was no increase
in cerebellar Amp levels in the A/A group. Second, Amp effects on NT/N
mRNA and immediate early gene expression are dose dependent (Castel
et al., 1993; Wang and McGinty, 1995b). Thus a higher Amp
concentration in the A/A group is unlikely to lead to the observed
reduction in responsiveness of NT/N or c-fos gene
expression. Finally, PNU-101387G-pretreated rats also displayed
behavioral sensitization to Amp (U/A) although their plasma levels were
similar to those seen in vehicle pretreated rats (V/A). Hence, at least
the cross-sensitization between the D4 antagonist and Amp could not be
due to pharmacokinetic factors regulating Amp concentrations.
c-fos mRNA induction.
Acute blockade of D4
receptors by PNU-101387G induced c-fos mRNA expression in
layers V and VI of the IL/vPL cortex as seen previously (Merchant
et al., 1996b). Acute Amp also increased c-fos
mRNA in deep layers of the IL/vPL cortex. However, there appear to be
distinct differences with respect to c-fos induction by Amp
and PNU-101387G: 1) c-fos induction by acute Amp became refractory after repeated Amp administration but not after repeated PNU-101387G administration. 2) Preexposure to PNU-101387G did not
reduce the capacity of a subsequent PNU-101387G challenge to induce
c-fos mRNA levels. Thus Amp and PNU-101387G appear to target
distinct neuronal populations in the medial prefrontal cortex. These
data are relevant because they indicate that PNU-101387G blocked the
development of the neuroadaptive c-fos response to Amp not
by directly influencing c-fos gene expression in
Amp-responsive neurons, but by an indirect mechanism that may involve
cortical-cortical interactions. Further indirect support for the
possibility that the medial prefrontal cortex is the site at which
PNU-101387G exerts its blockade of Amp sensitization (behavioral and
associated genomic/biochemical responses) derives from 1) the
predominantly cortical localization of the D4 receptor (Mrzljak
et al., 1996; Ariano et al., 1997; Defagot
et al., 1997) and, 2) our previous results that peripheral
administration of PNU-101387G induces c-fos gene expression
only in the prefrontal cortex within the forebrain (Merchant et
al., 1996b). Regardless of the precise mechanism, changes in
c-fos regulation by Amp in the medial prefrontal cortex
indicates that this region participates in neuroadaptive postsynaptic
responses associated with behavioral sensitization to Amp and their
modulation by the D4 receptor. These data are in agreement with the
conclusions of Wolf et al. (1995), and Karler et
al. (1997) who demonstrated that the medial prefrontal cortex plays a critical role in Amp sensitization and its modulation by
D2-like receptors.
Compared to the deep layers, c-fos mRNA induction in the
superficial IL/vPL cortex showed several distinct characteristics: 1)
Amp, but not PNU-101387G, significantly increased c-fos mRNA expression, 2) pretreatment with PNU-101387G by itself reduced the
ability of the Amp challenge to increase c-fos mRNA levels and 3) perhaps due to this, the refractoriness to Amp was not blocked
by concurrent administration of PNU-101387G with Amp during pretreatment. The reasons underlying these distinctions in PNU-101387G effects remain unclear but indicate that dopamine tone in distinct layers of the IL/vPL cortex may be differentially modulated by the D4
receptor.
The neostriatum has been studied widely and proposed to be a critical
site involved in postsynaptic neuroadaptive responses underlying
behavioral sensitization to Amp. These studies generally have used a
significantly higher dose of Amp (e.g., >5 mg/kg for pretreatment and challenge) than the dose of 2 mg/kg, s.c., used in our
studies (Graybiel et al., 1990; Jaber et al.,
1995). At 2 mg/kg, acute Amp did not induce c-fos mRNA
levels in the dorsal striatum or nucleus accumbens but profoundly
increased c-fos mRNA expression in the medial prefrontal
cortex. Interestingly, a greater sensitivity of dopamine and glutamate
systems in the prefrontal cortex (compared to neostriatum) has been
observed also after repeated low doses of methamphetamine using a
dosing and withdrawal protocol almost identical to the one used in our
study (Stephans and Yamamoto, 1995). Thus the medial prefrontal cortex
is not only a critical site but also a more sensitive region to acute and neuroadaptive responses produced by Amp and other psychostimulants.
NT/N mRNA induction.
Another molecular alteration accompanying
behavioral sensitization to Amp and its blockade by PNU-101387G was a
reduction in the inducibility of NT/N mRNA expression in the nucleus
accumbens-shell by Amp. Direct injections of NT in the accumbens
antagonize acute locomotor stimulation produced by Amp (Ervin et
al., 1981). Additionally, immunoneutralization of accumbal NT
potentiates locomotor activity and rearing produced by methamphetamine
(Wagstaff et al., 1994). These data indicate that accumbal
NT may antagonize the behavioral stimulatory effects of Amp. Hence, a
reduction in the capacity of Amp to induce NT/N gene expression in the
NA-s raises the question whether the reduced NTergic tone (as indicated
by mRNA levels) may contribute to enhanced behavioral stimulation
(sensitization) produced by Amp challenge in the Amp-pretreated rats.
Studies measuring NT release in the accumbal shell accompanying
behavioral sensitization will help determine if there indeed is a
reduction in the activity of this pathway.
Acute Amp-induced NT/N mRNA expression involves activation of D1
receptors (Castel et al., 1993). Because Amp-pretreated rats showed an augmented extracellular dopamine concentration (our study)
and D1 receptor function appears to be enhanced by repeated psychostimulant administration (Terwilliger et al., 1990;
Wolf et al., 1993), it was surprising to observe a reduction
in the capacity of Amp to induced NT/N mRNA levels. The induction of accumbal NT-like immunoreactivity by Amp appears to involve activation of not only D1 dopamine but also glutamate receptors (Singh et al., 1990). Hence, it is likely that the reduction in Amp-induced NT/N gene expression is due to a decrease in the sensitivity of accumbal neurons to glutamate after withdrawal from chronic Amp as
demonstrated by White et al. (1995). Immunohistochemical
studies do not show D4 receptor expression in the accumbal sector.
Hence, one possible mechanism by which D4 blockade prevents the
neuroadaptive response of accumbal neurons may involve modulation of
the activity of prefrontal cortical efferents to accumbal shell. Our
recent unpublished results support this hypothesis since there appears to be an increase in glutamatergic drive on prefrontal cortical neurons
in Amp-sensitized rats and this effect is blocked by coadministration of the D4 antagonist during Amp pretreatment (K.M. Merchant, B.K. Yamamoto).
Extracellular dopamine concentrations.
Previous studies have
shown an increase in the capacity of Amp to induce extracellular
dopamine levels in the NA after extended withdrawal from repeated,
intermittent Amp treatment (reviewed by Kalivas and Stewart, 1991;
Robinson and Berridge, 1993, White and Wolf, 1991). In our study,
Amp-induced increases in extracellular dopamine concentration was
augmented in rats withdrawn for an intermediate time period (7 days)
from an Amp-pretreatment treatment; a protocol that also induced robust
behavioral sensitization. Paulson and Robinson (1995) failed to observe
behavioral sensitization or augmented dopamine release after 7-day
withdrawal. The apparent discrepancy may be due to vast differences in
pretreatment protocols (6 wk of escalating dosing by Paulson and
Robinson, 1995, vs. 5 days at 2 mg/kg/day in our study) or
challenge doses employed. Additionally, our study demonstrated the
augmentation in Amp-induced extracellular dopamine levels in the shell
but not the core; Paulson and Robinson (1995) did not distinguish
between the two sectors of the NA. The regional selectivity in
presynaptic dopamine system adaptations are in line with the limbic
system attributes of the accumbal shell neurons (Deutch et
al., 1993). The increase in dopamine terminal sensitivity to Amp
shown here is in complete agreement also with the results of Pierce and
Kalivas (1995) who showed that microinjections of Amp into the accumbal
shell, but not the core, of cocaine-pretreated rats produce behavioral
sensitization and augmented dopamine release.
Because dopamine transmission in the nucleus accumbens mediates
locomotor stimulatory effects of psychostimulants, it is believed that
the augmentation in Amp-induced dopamine release in Amp-pretreated animals may contribute to the behavioral sensitization seen in these
rats (Robinson and Berridge, 1993). Behavioral evaluation was not
carried out in our study during in vivo microdialysis. However, the pretreatment and challenge protocols were identical between the behavioral and microdialysis studies (including the vehicles used and the pH of each drug solution). Hence, it is likely
that the augmentation in Amp-induced extracellular dopamine concentration in Amp-pretreated rats was accompanied by and contributed to behavioral sensitization. This possibility is supported also by the
observation that cotreatment of PNU-101387G with Amp blocked both the
behavioral sensitization and the augmented dopamine response to the Amp
challenge in Amp-pretreated rats.
Role of D4 receptors in Amp sensitization.
The selectivity of
PNU-101387G for dopamine D4 receptors has been examined extensively and
reported by Merchant et al. (1996b). PNU-101387G shows
moderately high affinity for the rat and human D4 receptor
(Ki = 10 nM). However, its affinity
at other dopamine, serotonin, noradrenergic, GABA or glutamate, and
several neuropeptide receptors is more than 1.5 µM. It appears that
it is the selectivity for the D4 receptor that enables PNU-101387G to
produce behavioral, biochemical and physiological effects that are
distinct from nonselective blockers of the D2 class of receptors (Merchant et al., 1996b). Immunolabeling studies in nonhuman
primates and rats have shown the highest level of expression of the D4 receptor in cortical regions followed by discrete basal ganglia nuclei
(Mrzljak et al., 1996; Ariano et al., 1997;
Defagot et al., 1997). Hence, PNU-101387G may exert its
effects through prefrontal cortical D4 receptors to block behavioral,
biochemical and genomic responses produced by repeated Amp exposure.
The preceding statement is based on the known selectivity of
PNU-101387G for D4 receptors. However, as with any pharmacological
agent, it can be argued that the substrate for PNU-101387G is a protein
other than D4 that remains unidentified or untested.
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Summary and Conclusions |
Our study demonstrates that the medial prefrontal cortex, NA shell
and the dopamine D4 receptor play a critical role in behavioral, biochemical and genomic events produced by repeated, intermittent Amp
exposure. It is likely that the effects of the D4 receptor antagonist
are mediated by alterations in prefrontal cortical neuronal activity.
Our results simply show an association of the biochemical (augmentation
in Amp-induced dopamine release) and genomic responses (reduction in
Amp-induced c-fos and NT/N mRNA induction) to Amp-induced
behavioral sensitization. The blockade of both, the induction of
behavioral sensitization and biochemical/genomic responses by the D4
antagonist suggests a causal relationship between the neural
adaptations and behavior. However, future studies directly examining
the functional relationship between these parameters are required to
understand the contribution of the neuronal adaptations to behavioral
changes produced by Amp.
Behavioral sensitization to Amp is seen not only in rodents but also in
humans. Humans show sensitization in the form of precipitation of
drug-induced psychotic episodes at lower doses in former drug addicts
compared to naive individuals (reviewed by Lieberman et al.,
1997). Whether or not the above demonstrated ability of PNU-101387G to
block behavioral sensitization to Amp and accompanying biochemical and
genomic effects indicate antipsychotic activity of this agent remains
to be tested in clinical studies.
PNU-101387G, (S(-)-4-[4-[2-(isochroman-1-yl)ethyl]piperazin-1-yl]benzenesulfonamide);
amphetamine, Amp, NT/N, neurotensin/neuromedinn;
VTA, ventral tegmental
area;
LMA, locomotor activity;
IL, infralimbic cortex;
vPL, ventral
prelimbic cortex;
NA, nucleus accumbens;
NA-s, nucleus accumbens-shell;
NA-c, nucleus accumbens-core.
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Abstract
Introduction
