Introduction

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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.

	Materials and Methods

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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.

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).

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.

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

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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.

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).

View larger version (20K): [in this window] [in a new window]   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.

View larger version (28K): [in this window] [in a new window]   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.

View larger version (94K): [in this window] [in a new window]   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.

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).

View larger version (59K): [in this window] [in a new window]   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.

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.

View larger version (163K): [in this window] [in a new window]   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.

View larger version (42K): [in this window] [in a new window]   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.

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).

View larger version (28K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion Conclusions References

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.

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.

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.

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.

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 (K_i = 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.

	Summary and Conclusions

Top Abstract Introduction Materials & Methods Results Discussion Conclusions References

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.