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BEHAVIORAL PHARMACOLOGY

Effects of Muscarinic M₁ Receptor Blockade on Cocaine-Induced Elevations of Brain Dopamine Levels and Locomotor Behavior in Rats

Psychobiology (G.T., A.L.E., T.A.K., L.M.E., B.L.C., J.L.K.) and Medicinal Chemistry (A.H.N.) Sections, Medications Discovery Research Branch, Department of Health and Human Services, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland

Received for publication December 1, 2006
Accepted January 23, 2007.

	Abstract

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Cholinergic muscarinic systems have been shown to influence dopaminergic function in the central nervous system. In addition, previous studies of benztropine analogs that inhibit dopamine uptake and show antagonism at muscarinic receptors show these drugs to be less effective than cocaine in producing its various prototypic effects such as locomotor stimulation. Because previous pharmacological studies on these topics have used nonselective M₁ antagonists, we examined the interactions of preferential M₁ muscarinic antagonists and cocaine. Dose-dependent increases in extracellular levels of dopamine in selected brain areas, the nucleus accumbens (NAc) shell and core, and the prefrontal cortex, were produced by cocaine but not by the preferential M₁ antagonists telenzepine and trihexyphenidyl. When administered with cocaine, however, both M₁ antagonists dose-dependently increased the effects of cocaine on dopamine in the NAc shell, and these effects were selective in that they were not obtained in the NAc core or in the prefrontal cortex. Telenzepine also increased locomotor activity, although the effect was small compared with that of cocaine. The locomotor stimulant effects of trihexyphenidyl, in contrast, approached those of cocaine. Telenzepine attenuated, whereas trihexyphenidyl enhanced the locomotor stimulant effects of cocaine, with neither drug facilitating cocaine-induced stereotypy. The present results indicate that preferential antagonist effects at muscarinic M₁ receptors do not uniformly alter all of the effects of cocaine, nor do they explain the differences in effects of cocaine and benztropine analogs, and that the alterations in dopamine levels in the NAc shell do not predict the behavioral effects of the interactions with cocaine.

Among the many molecules that have been synthesized in research on medications for cocaine abuse, analogs of benztropine (BZT) are of interest because they have a high affinity for the DA transporter (Newman et al., 1994; Newman and Kulkarni, 2002), while showing reduced cocaine-like activities in experimental animals (Katz et al., 1999, 2001), including reduced reinforcing effects (Woolverton et al., 2000). Like the parent compound, many of the BZT analogs have affinity for acetylcholine muscarinic receptors. Because an increase in DA transmission in striatal areas is thought to play an important role in the behavioral effects of cocaine, diminished DA transmission in these areas due to blockade of M₁ receptors, as described above, might explain the reduced cocaine-like effects of BZT analogs. However, systemically administered nonselective muscarinic antagonists (atropine and scopolamine) increased, rather than decreased, the behavioral effects of cocaine (Scheckel and Boff, 1964; Wilson and Schuster, 1973; Katz et al., 1999).

Although the role of muscarinic actions in the reduced cocaine-like effects of BZT analogs has been the subject of several studies, the role for muscarinic receptor subtypes has not been studied. Because several of the BZT analogs have preferential affinity for M₁ muscarinic subtypes (Katz et al., 2004), the aim of the present study was to further investigate how preferential muscarinic M₁ blockade alters the behavioral effects of cocaine in rats, and to compare those effects with the alterations in the neurochemical effects of cocaine. To this end, we compared some behavioral and neurochemical effects of cocaine alone and after pretreatment with preferential antagonists of M₁ muscarinic receptors. Although many of the studies of effects of muscarinic antagonists on DA transmission have used local injections of the muscarinic drugs in the central nervous system, we administered muscarinic antagonists systemically to more closely replicate the effects of BZT analogs.

Telenzepine (TZP) and trihexyphenidyl (TXP) are reported to have preferential activity at M₁ receptors compared with other muscarinic subtypes (Eltze et al., 1985; Doods et al., 1987; Bymaster et al., 1993). Previous studies in mice showed that selected doses of TXP reduced both the elevations in striatal DA levels and the place conditioning produced by methamphetamine, although curiously, it did not alter those same effects when produced by cocaine (Shimosato et al., 2001, 2003). If the interactions observed with methamphetamine and TXP were extended to cocaine with more extensive comparisons of doses, they might account for the lack of cocaine-like effects of BZT analogs, and they might help better understand the previous studies with the nonselective M₁ antagonists. Thus, in the present investigation, we studied interactions of a broad range of dose combinations of M₁ antagonists and cocaine, using DA microdialysis and locomotor activity in rats. Because the behavioral relevance of changes in DA neurotransmission depends critically on the dopaminergic terminal area, we examined the effects in the NAc shell, the NAc core, and the medial prefrontal cortex.

	Materials and Methods

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Subjects. Male Sprague-Dawley rats (Taconic, Germantown, NY), experimentally naive at the start of the study and weighing 300 to 350g, were doubly housed and had free access to food and water. All rats were housed in a temperature- and humidity-controlled room and were maintained on a 12-h light/dark cycle (lights were on from 7:00 AM to 7:00 PM). Experiments were conducted during the light phase.

Subjects used in this study were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care International, and all experimentation was conducted in accordance with the guidelines of the Animal Care and Use Committee of the Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, and the Guide for Care and Use of Laboratory Animals (National Research Council, 1996).

Binding Studies. Affinity for the DA transporter was assessed by determining displacement of [³H]WIN 35,428 (PerkinElmer Life and Analytical Sciences, Boston, MA) binding to tissue from male Sprague-Dawley rats (Taconic Farms, Germantown, NY). Striatum was dissected and frozen. On the day of the assay, the striatum was homogenized in 30 volumes of ice-cold sucrose-phosphate buffer (10 mM NaHPO₄ and 0.32 M sucrose, pH 7.4) and centrifuged at 20,000g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed once more by resuspension in ice-cold buffer and centrifuged at 20,000g for 10 min at 4°C. After the second wash, the pellet was resuspended to give 10 mg/ml wet weight, final concentration. Assays were conducted in 0.5 ml of the sucrose-phosphate buffer, at pH 7.4 on ice. Each tube contained buffer, one of 14 concentrations of the displacer drug (including zero), 1.0 mg of tissue, and [³H]WIN 35,428. The final concentration of [³H]WIN 35,428 was 0.5 nM. Nonspecific binding was determined by the addition of cocaine HCl at a 100 µM final concentration. Triplicate tubes were assayed. The reaction was started with the final addition of [³H]WIN 35,428, after which the contents were allowed to incubate for 120 min on ice. The incubation ended with the addition of ice-cold buffer and rapid filtration through a Brandel R48 (Brandel Inc., Gaithersburg, MD) filtering manifold, using GF/B glass fiber filter paper (Whatman, Clifton, NJ) that was presoaked in 0.05% polyethyleneimine (PEI) in water to reduce nonspecific binding. The filters were washed twice with 5 ml of ice-cold buffer, and then they were transferred to scintillation vials. Three milliliters of scintillation cocktail (Beckman Ready Safe; Beckman Coulter, Fullerton, CA) was added to the vials, which were counted the following day using a Beckman 6000 liquid scintillation counter (Beckman Coulter).

Affinity for M₁ muscarinic receptors was assessed by determining displacement of [³H]pirenzepine (PerkinElmer Life and Analytical Sciences, Boston, MA) binding to membranes from frozen rat brains excluding cerebellum (Taconic Farms). On the day of the assay, tissue was thawed in 30 volumes of ice-cold buffer (10 mM Tris-HCl and 320 mM sucrose, pH 7.4) and homogenized with a Brinkman Polytron homogenizer (Instruments, Westbury, NY) in a volume of 10 ml/g tissue. The homogenate was centrifuged at 1000g for 10 min at 4°C. The supernatant was then centrifuged at 10,000g for 20 min at 4°C. The resulting pellet was resuspended in a volume of 10 mM Tris buffer, pH 7.4, to give 200 mg/ml wet weight, final tissue concentration. Assays were conducted in 0.5 ml of buffer (10 mM Tris-HCl and 5 mM MgCl₂) in a 37°C water bath. Each tube contained 15 nM [³H]pirenzepine, 20 mg of tissue, and one of 14 concentrations (including zero) of displacer. The final concentration of [³H]pirenzepine was 0.75 nM. Quinuclidinyl benzilate, 100 µM final concentration, was used to determine nonspecific binding. Triplicate tubes were assayed. The reaction was started with the addition of the tissue, and the tubes were incubated for 60 min. The incubation was terminated by the addition of 5 ml of ice-cold buffer (10 mM Tris-HCl, pH 7.4) and rapid filtration through Whatman GF/B glass fiber filter paper (presoaked in 0.5% PEI) using a Brandel cell harvester. The filters were washed twice with 5 ml of ice-cold buffer and transferred to scintillation vials to which 3 ml of Beckman Ready Safe was added. The vials were counted the following day using a Beckman 6000 liquid scintillation counter.

Affinity for M₂, M₃, M₄, and M₅ muscarinic receptors was estimated by assessing displacement of [³H]N-methylscopolamine binding to each of the corresponding human recombinant muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Assays were modified from those reported by Buckley et al. (1989), and they were conducted in duplicate with two replicates using [³H]N-methylscopolamine, at 0.5, 0.2, 0.2, and 0.2 nM to label, respectively, at M₂ through M₅ muscarinic receptor sites. Specific binding was defined with 1.0 µM methylscopolamine bromide in each of the assays. Historical K_D values for [³H]N-methylscopolamine at these sites are 0.29, 0.14, 0.09, and 0.2 nM, respectively, at the M₂ through M₅ muscarinic receptor subtypes. Cells were thawed in phosphate-buffered saline. Each tube received 50 µl of drug or vehicle, 50 µl of [³H]N-methylscopolamine, 400 µl of cell suspension, and one of three concentrations (1, 100, and 10,000 nM) of displacer. The binding reaction was initiated with the addition of the cells, and the contents were incubated at 25°C for 60 min. The reaction was terminated by rapid vacuum filtration of the tube contents onto presoaked (0.3% PEI) Whatman Topcount GF/B filters. After five rinses with ice-cold 50 mM NaCl, the radioactivity trapped on the filters was assessed using liquid scintillation counting. Under these conditions, specific binding ranged from greater than 80% of total at the lowest concentration of displacer to less than 2% of total at the highest concentration studied. The assays were conducted by Novascreen (Hanover, MD) through a contract with the National Institute on Drug Abuse (Baltimore, MD).

In Vivo Microdialysis. Under a mixture of ketamine and xylazine (60.0 and 12.0 mg/kg i.p., respectively) anesthesia, rats were placed in a stereotaxic apparatus, the skull was exposed, and a small hole was drilled to expose the dura. Rats were then randomly implanted in the right or left brain side with a concentric dialysis probe (see below) aimed at the NAc shell or core, or at the medial PFCX, as described previously (Tanda et al., 1997, 2005; also see Fig. 1 for placements). Coordinates were from the rat brain atlas by Paxinos and Watson (1987) (uncorrected coordinates: shell: A, +2.0; L, ±1.1; and V, 7.9; core: A, +1.6; L, ±1.9; and V, 7.7; and PFCX: A, +3.5; L, ±0.6; and V, 5.0; A and L, millimeters from bregma; V, millimeters from dura). After the surgery rats were allowed to recover overnight in hemispherical CMA-120 cages (CMA/Microdialysis, Solna, Sweden) equipped with overhead fluid swivels (Instech Laboratories Inc., Plymouth Meeting, PA) for connections to the dialysis probes.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Forebrain sections, redrawn from Paxinos and Watson (1987), showing the limits of the positions of the dialyzing portions of the microdialysis probes (superimposed rectangles). The anterior coordinate (measured from bregma) is indicated on each section. CPU, caudate putamen; Co, NAc core; Sh, NAc shell.

Concentric dialysis probes were prepared with AN69 fibers (Hospal Dasco, Bologna, Italy). In brief, two 4-cm pieces of silica-fused capillary tubes (the inlet and outlet tubing of the probes) were inserted into a 6-mm capillary (0.25-mm external diameter) dialyzing fiber (closed by a drop of glue on the other side), with the inlet tubing set at approximately 0.1 mm from the closed end of the fiber, and the outlet set at 2.0 (NAc probes) or 3.0 mm (PFCX probes) from the inlet tip. The open end of the dialysis membrane was then glued, and the protruding two silica-fused tubings were inserted and glued into a 22-gauge stainless steel needle (2.4 mm in length). The needle was attached to a CMA/10 clip (CMA/Microdialysis), and the needle and clip were mounted in a stereotaxic holder. The exposed dialyzing surface of the fibers, i.e., not covered by glue, was limited to the lowest 2.0-mm (NAc) or 3.0-mm (PFCX) portion of the probes.

Experiments were performed on freely moving rats in the same hemispherical cages in which they recovered from surgery. Approximately 22 to 24 h after implant, probes were connected to fluid swivels (375/D/22QM; Instech Laboratories Inc.), and Ringer's solution (147.0 mM NaCl, 2.2 mM CaCl₂, and 4.0 mM KCl) was delivered by a 1.0-ml syringe, operated by a BAS Bee syringe pump controller (BAS Bioanalytical Systems, West Lafayette, IN), through the dialysis probes at a constant flow rate of 1 µl/min. Collection of dialysate samples started after 30 min, and 10-µl samples were taken every 10 min and immediately analyzed, as detailed below. After stable DA values (less than 10% variability) were obtained for at least three consecutive samples (typically after approximately 1 h), rats received a pretreatment injection (M₁ antagonist or saline), and after 20 more minutes, they were treated with cocaine or saline. Rats were used only once and received only one pretreatment/treatment drug combination. Throughout the study, contemporaneous saline pretreatment controls were conducted with TXP- or TZP-pretreated subjects to ensure appropriate comparisons for statistical analysis. In addition, the effects of 3.0 mg/kg cocaine in the NAc shell were redetermined in a new group of subjects after the interactions had been studied to ensure reliability of results.

Dialysate samples were injected without purification into a high-performance liquid chromatography apparatus equipped with an MD 150- x 3.2-mm column, particle size 3.0 µm (ESA, Chelmsford, MA) and a coulometric detector (5200a Coulochem II; ESA) to quantify DA. The oxidation and reduction electrodes of the analytical cell (5014B; ESA) were set at +125 and –125 mV, respectively. The mobile phase, containing 100 mM NaH₂PO₄, 0.1 mM Na₂EDTA, 0.5 mM n-octyl sulfate, and 18% (v/v) methanol (pH adjusted to 5.5 with Na₂HPO₄), was pumped by an ESA 582 solvent delivery module at 0.60 ml/min. Assay sensitivity for DA was 2 fmol/sample.

At the end of the experiment, rats were euthanatized by pentobarbital overdose. Their brains were removed and left to fix in 4% formaldehyde in saline solution. Brains were then cut on a Vibratome Plus 1000 (The Vibratome Company, St. Louis, MO) in serial coronal slices (orientation as per Paxinos and Watson, 1987) to identify the location of the probes. In all the experiments, the location of the probes was verified. Figure 1 schematically shows typical locations of the dialyzing portion of the probes implanted in each region. The sections are redrawn (Paxinos and Watson, 1987), and the anterior coordinates (measured from bregma) for each brain area are indicated. Only the experiments in which the probes were located in these areas have been used herein.

Locomotor Activity. Subjects were placed in square (40- x 40-cm) acrylic chambers that were placed inside monitors (Omnitech Electronics, Columbus, OH) equipped with light-sensitive detectors (photocells) spaced 2.5 cm apart along two perpendicular walls. Mounted on the opposing walls were infrared light sources directed at the photocells. One count of horizontal activity was registered each time the photocell was activated by interruption of the light source. Subjects were allowed to habituate for 120 min before injections were administered. At that point, injections of either saline, TXP (0.3–3.0 mg/kg), or TZP (0.1–3.0 mg/kg) were administered i.p., and the subjects were immediately returned to the apparatus. Cocaine (1.0–10.0 mg/kg) was injected 20 min later, and subjects were again immediately returned to the apparatus. Horizontal locomotor counts were tabulated each 10 min for the next 180 min. Each dose was studied in six subjects, and subjects were used only once.

Stereotypy. Because the effects of the drugs and drug combinations on locomotor activity could have been attributed to interfering stereotyped behaviors, we conducted a study of the induction of various stereotyped behaviors in a separate group of subjects. The subjects were placed in square (40- x 40-cm) acrylic chambers. They were allowed to habituate for 120 min, and then they were injected with either saline, TXP (0.3 or 1.0 mg/kg s.c.), or TZP (0.3 or 1.0 mg/kg s.c.), followed 20 min later by cocaine (3.0 or 10.0 mg/kg i.p.) or saline. Because cocaine effects were most pronounced within the first 30 min after injection, only those data are presented. Behavior of each subject was observed for 1 min, every 10 min over the course of the next 30 min (i.e., each subject was observed three times at 10-min intervals).

The behavior of each subject was rated as belonging within one of the 10 categories of a scale originally described by Kalivas et al. (1988). The rater was blind to the treatments. The categories were as follows: 1) asleep or still; 2) inactive, grooming, mild licking; 3) locomotion (all four feet move in 10 s), rearing or sniffing (3-s duration); 4) any combination of two: locomotion, rearing, or sniffing; 5) continuous sniffing for 10 s with neither locomotion nor rearing; 6) continuous sniffing for 10 s with locomotion or rearing; 7) patterned sniffing for 5 s; 8) patterned sniffing for 5 s; 9) continuous gnawing; and 10) bizarre dyskinetic movements or seizures. As can be seen from their description, categories lower than 4 do not involve behaviors generally considered as stereotyped. Behaviors falling into category 4 can be considered a low intensity of stereotyped behavior, whereas those behaviors in categories 5 and above can be considered frank stereotypy.

Drugs. The drugs tested were as follows: telenzepine diHCl (TZP; Sigma-Aldrich, St Louis, MO), trihexyphenidyl HCl (TXP; Sigma-Aldrich), and (–)-cocaine HCl (Sigma-Aldrich and National Institute on Drug Abuse). Drugs were dissolved in 0.9% NaCl and were injected s.c. (TZP and TXP) or i.p. (cocaine) in a volume of 1.0 ml/kg. In the binding assays (Table 1), we also tested the following BZT analogs (Newman et al., 1994): AHN 1-055, 4'-4''-diCl-BZT, and 4-Cl-BZT. The hypothesis of the study was that antagonism at M₁ muscarinic receptors would reduce the neurochemical effects of cocaine; however, initial studies with 0.3 mg/kg TZP showed an increase in neurochemical effects. Therefore, subsequent studies confirmed a dose-dependent enhancement of the effects of an intermediate dose of cocaine, and we selected this intermediate dose for more extensive investigation of the dose effects of TZP, with the assumption that any antagonism would be more readily obtained against a submaximal dose of cocaine. Because differences emerged in the behavioral effects of the M₁ antagonists, a larger range of doses was tested in the studies of locomotor activity.

Data Analysis. All binding data were analyzed using a nonlinear, least-squares regression analysis (GraphPad Prism Software Inc., San Diego, CA), with K_i values calculated from IC₅₀ values using the equation of Cheng and Prusoff (1973) and historical K_D values of the radioligand. The data from the studies of displacement of [³H]pirenzepine by telenzepine or trihexylphenidyl from rat brain membranes were assessed for whether they better fit a one- or two-site model. Because IC₅₀ values for binding to M₂, M₃, M₄, and M₅ receptors were determined from three concentrations of nonradioactive cold compound, the derived binding constants are considered estimates and presented without error terms.

In the microdialysis study, results are expressed as a percentage of basal DA values, which were calculated as means of three consecutive samples (differing by no more than 10%) immediately preceding the first drug or vehicle injection. All results are presented as group means ± S.E.M. Statistical analysis (STATISTICA software; StatSoft, Tulsa, OK) was carried out using one- or two-way analyses of variance (ANOVA) for repeated measures over time applied to the data obtained from serial assays of dialysate DA normalized as percentage of basal values of each group. Significant results were subjected to post hoc Tukey's tests. Statistical analysis of differences in basal DA values (femtomoles per 10-µl sample ± S.E.M.) between different experimental groups and brain areas was carried out with one-way ANOVA. Changes were considered to be significant when p < 0.05. Sample size and basal DA values, expressed as femtomoles per sample ± S.E.M., for each microdialysis experimental group are indicated in the corresponding figure legends.

For the locomotor activity study, results were expressed as horizontal counts (instances of photocell activation). Two- or three-way ANOVAs were performed on each test drug such that the effects of dose and time were assessed. Tukey's honestly significant difference post hoc tests provided pairwise comparison information.

The categorical descriptions of the Kalivas et al. (1988) scale represent a progression of various different effects, and they are essentially nominal in nature (particularly at the lower numbered categories). Therefore, the data were recorded as frequencies per category and not subjected to mathematical operations across categories. Each of the six subjects was observed three times during the first 30 min after injection, and those three frequencies were used to obtain a standard error. There were few, if any, frequencies of category 1 behaviors; category 2 results were essentially the inverse of the effects on locomotor activity; and category 3 results essentially replicated the results obtained in the apparatus designed to measure locomotor activity. Therefore, only frequencies of behavior defined as falling in category 4 and greater are presented. Because there were no instances of category 4 behaviors at 3.0 mg/kg, the frequencies of category 4 behaviors obtained with cocaine alone at the 10-mg/kg dose were compared with frequencies obtained for cocaine with the doses of M₁ antagonists by a Fisher's exact probability test. Because there were no instances of frank stereotypy (behaviors in categories 5–8), no analysis of data from these categories was necessary.

	Results

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Receptor Binding Studies. Both TZP and TXP displaced [³H]pirenzepine from rat brain membranes with high affinity (Table 1), and the data were better fit to a one-site than a two-site model. TZP had approximately 5- to 20-fold higher affinity at M₁ than M₂, M₃, M₄, or M₅ receptors. In addition, it had more than 4 orders of magnitude greater affinity at M₁ muscarinic receptors than it did the DA transporter. TXP was less selective than TZP, showing approximately 5-fold preferential affinity for M₁ over M₂ receptors, but with affinity only approximately 2-fold higher than (M₃ and M₅) or equal to (M₄) M₁ affinity at the other muscarinic receptors. Like TZP, its affinity for the DA transporter was much lower (more than 3 orders of magnitude) than its affinity for M₁ muscarinic receptors. The BZT analogs generally had preferential affinity for M₁ muscarinic receptors over the other subtypes, with a maximum of approximately 5-fold selectivity; however, for some individual comparisons (e.g., M₁ versus M₃ affinities for 4-Cl-BZT), there was no selectivity. The values for affinities at M₂, M₃, M₄, and M₅ receptors and their ratios should be considered estimates only, as a result of the assay conditions (see Materials and Methods).

Microdialysis Studies. Neither TZP (0.3, 1.0, and 3.0 mg/kg s.c.) nor TXP (0.3 and 1.0 mg/kg s.c.) when administered alone had significant effects on levels of extracellular DA in the NAc shell during the entire 180-min observation period (data not shown). For TZP, the ANOVA indicated a main effect of time (F_15,135 = 3.76; p < 0.01) with nonsignificant (N.S.) main effects of dose and dose x time interactions. TZP was similarly inactive in the NAc core (ANOVA: N.S. main effects of dose, time, and their interaction) and in the PFCX (ANOVA: N.S. main effects of dose, time, and their interaction). For TXP, separate two-way ANOVAs on effects in the NAc shell and core indicated no significant main effects of either treatment or time, nor were there significant interactions of the two. In contrast to the muscarinic antagonists, cocaine (1.0–10 mg/kg i.p.) produced dose-related increases in extracellular DA in the shell of the NAc (Fig. 2A–C, filled symbols; note scale differences for ordinates). The maximal effects of cocaine at the doses studied were obtained within 30 min after injection. These effects of cocaine are similar to those shown previously in this and other laboratories (Kuczenski et al., 1991; Tanda et al., 1997, 2005), and a replication of the effects of 3.0 mg/kg cocaine in the NAc shell showed no significant differences in effects (F_1,12 = 0.024; p = 0.880).

View larger version (52K): [in this window] [in a new window]   Fig. 2. Time courses for the effects of cocaine (1.0, 3.0, or 10.0 mg/kg i.p., administered at time 0) with saline or TZP (0.3 mg/kg s.c.) administered 20 min before. Extracellular levels of DA in dialysates from the NAc shell (top), NAc core (middle), and PFCX (bottom) were examined. B, E, and H, effects of pretreatment with increasing doses of TZP on cocaine (3 mg/kg)-stimulated DA extracellular levels in dialysates from the NAc shell (B), NAc core (E), and PFCX (H). Each point represents means (with vertical bars representing S.E.M.) of the amount of DA in 10-min dialysate samples, expressed as percentage of basal values, uncorrected for probe recovery, with the exception of the first points, which represent the basal values determined from the mean of three 10-min samples collected immediately before the first injection. Values for DA concentration are represented on the figure at the point corresponding to the end of the sampling period. The sample sizes with basal DA values expressed as femtomoles/sample ± S.E.M. in parentheses were as follows: in the NAc shell, four (57.9 ± 7.31), six (55.7 ± 8.50), and five (58.0 ± 8.20) for saline with cocaine doses of 1.0, 3.0, and 10.0 mg/kg, respectively; four (56.1 ± 9.57), four (47.1 ± 6.75), and four (62.9 ± 7.07) for TZP (0.3 mg/kg), with cocaine doses of 1.0, 3.0, and 10.0 mg/kg, respectively; and four (56.8 ± 11.8), four (47.1 ± 6.75), seven (66.9 ± 11.2), and five (46.4 ± 8.06) for TZP (0.1, 0.3, 1.0, and 3.0 mg/kg, respectively), with 3.0 mg/kg cocaine; in the NAc core, four (61.8 ± 8.75), six (59.8 ± 6.40), and six (50.7 ± 6.55) for saline with cocaine doses of 1.0, 3.0, and 10.0 mg/kg, respectively; four (55.0 ± 8.21), four (38.9 ± 3.75), and four (45.7 ± 9.1) for TZP (0.3 mg/kg), with cocaine doses of 1.0, 3.0, and 10.0 mg/kg, respectively; and four (38.9 ± 3.75), eight (49.5 ± 7.08), and four (48.2 ± 8.91) for TZP at doses of 0.3, 1.0, and 3.0 mg/kg, respectively, with cocaine (3.0 mg/kg); in the PFCX, four (13.6 ± 0.92), five (15.1 ± 3.08), and five (12.3 ± 0.97) for saline, with cocaine doses of 1.0, 3.0, and 10.0 mg/kg, respectively; four (13.6 ± 3.42), four (15.4 ± 1.20), and six (11.7 ± 1.13) for TZP (0.3 mg/kg), with cocaine doses of 1.0, 3.0, and 10.0 mg/kg respectively; and four (15.4 ± 1.20), four (8.21 ± 1.07), and four (9.64 ± 0.68) for TZP at doses of 0.3, 1.0, and 3.0 mg/kg, respectively, with cocaine dose of 3.0 mg/kg.

The alteration in the effects of cocaine (3.0 mg/kg) on DA levels in the NAc shell depended on TZP dose (Fig. 2B). The ANOVA indicated main effects of TZP dose (F_3,21 = 5.54; p < 0.01), time (F_12,252 = 142; p < 0.01), and a dose x time interaction (F_36,252 = 3.18; p < 0.05). The effect was absent at 0.1 mg/kg TZP, and it was most pronounced at 0.3 mg/kg. In contrast, TZP did not affect the actions of cocaine on DA in the microdialysis experiments in the NAc core (Fig. 2E) or the PFCX (Fig. 2H). A two-way ANOVA on effects in the NAc core indicated a significant main effect of time (F_15,270 = 67.4; p < 0.05) and N.S. effects of TZP dose and the dose x time interaction. The corresponding two-way ANOVA on effects in the PFCX also indicated a significant main effect of time (F_15,210 = 88.4; p < 0.05), without significant effects of either TZP dose or a dose x time interaction.

When TXP was administered 20 min before cocaine injection, the lower 0.3-mg/kg dose of TXP did not significantly modify the effects of 3.0 mg/kg cocaine on DA levels in the NAc shell (Fig. 3A). Two-way ANOVA showed a significant effect of time (F_12,96 = 102; p < 0.05) and N.S. effects of pretreatment and pretreatment x time interaction. The 1.0-m/kg dose of TXP enhanced the effects of cocaine at 3.0 and 10 mg/kg i.p. (Fig. 3, A and C) in the NAc shell. A three-way ANOVA indicated main effects of cocaine dose (F_2,23 = 45.3; p < 0.01), TXP pretreatment (F_1,23 = 11.3; p < 0.01), and time (F_12,276 = 58.6; p < 0.01); there were also significant interactions of cocaine dose x time (F_24,276 = 21.8; p < 0.01), cocaine dose x TXP pretreatment (F_2,23 = 4.7; p < 0.02), pretreatment x time (F_12,276 = 3.2; p < 0.01), and cocaine dose x TXP pretreatment x time (F_24,276 = 1.7; p < 0.05). In contrast, no significant alteration of the effects of either 3.0 or 10.0 mg/kg cocaine was produced by TXP in the core of the NAc (Fig. 3, B and D). A two-way ANOVA on the effects of 0.3 mg/kg TXP and 3.0 mg/kg cocaine indicated a significant effect of time (F_12,96 = 36.1; p < 0.05) but N.S. effects of treatment and treatment x time interaction. A three-way ANOVA indicated significant main effects of cocaine dose (F_2,23 = 79.4; p < 0.01) and time (F_12,276 = 103.84; p < 0.01) and a N.S. main effect of TXP pretreatment (F_1,23 = 0.0006; N.S.); there was also a significant interaction of cocaine dose x time (F_24,276 = 23.1; p < 0.01) and N.S. interactions of cocaine dose x TXP pretreatment (F_2,23 = 0.11; N.S.), TXP pretreatment x time (F_12,276 = 0.83; N.S.), and cocaine dose x TXP pretreatment x time (F_24,276 = 0.37; N.S.).

View larger version (40K): [in this window] [in a new window]   Fig. 3. Time courses for the effects of cocaine (3 and 10 mg/kg administered at time = 0) injected i.p. 20 min after s.c. saline or increasing doses of s.c. TXP on induced elevations of DA in dialysates from the NAc shell (A and C), or from the NAc core (B and D). The sample sizes (with basal DA values in femtomoles/sample ± S.E.M.) were as follows: in the NAc shell, six (55.7 ± 8.50), five (64.3 ± 7.58), and four (41.4 ± 6.03) for saline or TXP at 0.3 and 1.0 mg/kg, respectively, with cocaine at 3.0 mg/kg; five (58.0 ± 8.20) and six (40.4 ± 7.75) for saline or TXP at 1.0 mg/kg, respectively, with cocaine at 10 mg/kg; in the NAc core, six (59.8 ± 6.40), five (64.6 ± 4.22), and four (46.4 ± 4.61) for saline or TXP at 0.3 and 1.0 mg/kg, respectively, with cocaine at 3.0 mg/kg; six (50.7 ± 6.55) and five (61.4 ± 7.69) for saline or TXP at 1.0 mg/kg, respectively, with cocaine at 10.0 mg/kg. All other details are as shown in Fig. 2.

Locomotor Activity. TZP (0.1–3.0 mg/kg, administered s.c. 20 min before saline) increased locomotor activity (Fig. 4A), although the effects were small compared with those of cocaine (see below). An ANOVA indicated significant effects of dose (F_4,425 = 3.54; p = 0.02), time (F_17,425 = 12.5; p < 0.001), and a dose x time interaction (F_68,425 = 1.88; p < 0.001). Greater increases in locomotor activity were obtained with TXP (0.3–3.0 mg/kg, administered s.c. 20 min before saline; Fig. 4B). The ANOVA for these effects indicated significant effects of dose (F_3,340 = 10.9; p < 0.001), time (F_17,340 = 6.49; p < 0.001), and their interaction (F_51,340 = 1.57; p = 0.011). Cocaine (1.0–10 mg/kg, administered i.p. 20 min after saline) also increased locomotor activity in a dose-related manner (Fig. 4C). The ANOVA for these effects also indicated significant effects of dose (F_3,340 = 4.80; p < 0.011), time (F_17,340 = 12.3; p < 0.001), and their interaction (F_51,340 = 3.35; p < 0.001).

View larger version (62K): [in this window] [in a new window]   Fig. 4. Time courses for effects of TZP (0.1–3.0 mg/kg), TXP (0.3–3.0 mg/kg), or cocaine (1.0–10.0 mg/kg) alone and in combination on locomotor activity in rats. TZP, TXP, or saline was administered s.c. 20 min before i.p. injections of cocaine or cocaine-vehicle (saline), and locomotor activity was assessed immediately thereafter. Each point represents average locomotor activity (in counts per minute) for six subjects during a 10-min period, with vertical bars representing S.E.M. Cumulative counts are represented on the figure at the point corresponding to the end of the sampling period. In A, B, and C, the filled points represent the effects of two vehicle injections shown in each panel for comparisons, whereas in D through I, the filled points represent the effects of cocaine injected 20 min after saline. Open points represent effects of the increasing doses of TZP, TXP, or cocaine alone or in combination as indicated by the keys.

Administration of TXP (0.3–3.0 mg/kg) enhanced the effects of cocaine on locomotor activity, which was apparent at all three of the cocaine doses (Fig. 4, G–I). A three-way ANOVA indicated main effects of cocaine dose (F_3,86 = 21.3; p < 0.001), TXP pretreatment (F_3,86 = 26.4; p < 0.001), and time (F_17,86 = 30.4; p < 0.001). In addition, there were significant interactions of cocaine dose x time (F_51,86 = 4.11; p < 0.001), TXP dose x time (F_51,86 = 3.26; p < 0.001), and all three variables (F_153,86 = 1.57; p = 0.0108) but a N.S. cocaine dose x TXP dose interaction.

Stereotypy. At the doses of cocaine studied, there was no frank stereotypy (behaviors in categories greater than 4), nor were there behaviors in categories greater than 4 at combinations of cocaine with either of the M₁ antagonists (Table 2). Thus, neither M₁ antagonist showed any evidence of potentiating cocaine-induced stereotypy (Table 2). At 10 mg/kg, cocaine produced a N.S. increase (Fisher's exact probability test; p < 0.075) in the frequency of category 4 behaviors (any combination of two instances of locomotion, rearing, sniffing) from a value of 17% after vehicle to 50% (Table 2). TZP (0.3 and 1.0 mg/kg) decreased the frequency of category 4 behavior (Fisher's exact probability test; p < 0.007 and p = 0.001, respectively) when given before the 10-mg/kg dose of cocaine. TXP treatment (0.3 and 1.0 mg/kg) produced a less pronounced trend toward a decrease in the frequency of category 4 behaviors after the 10-mg/kg dose of cocaine (Table 2), which was not statistically significant.

View this table: [in this window] [in a new window]   TABLE 2 Effects of cocaine on the frequencies of occurrences of behaviors belonging to categories of stereotyped behaviors as described by Kalivas et al. (1988) Frequencies are expressed as percentages of occasions in which a behavior falling into that category occurred.

High doses of stimulant drugs elicit stereotypy that can interfere with ambulation, and TZP increased the effects of cocaine on extracellular DA levels in the NAc shell, which could render low doses of cocaine pharmacologically equivalent to higher doses. Thus, the decreases in effects of cocaine on locomotor activity produced by TZP could have been the result of increased effects of cocaine producing stereotypy, and thereby consistent with the effects on extracellular DA levels. Figure 5A shows the increases in the effects of cocaine on DA levels produced by TZP during the first 30 min after cocaine injection. Also shown is the decrease in effects of cocaine on locomotor activity (Fig. 5B) and frequency of category 4 behaviors in the stereotypy scale (Fig. 5C) for the first 30 min after treatment. At a cocaine dose alone that produced little behavior on the stereotypy scale (3.0 mg/kg), or a dose that produced a nonsignificant increase in category 4 behaviors (10 mg/kg), there was no potentiation of these effects by TZP, and no frank stereotypy (categories above 4) at any dose combination (Table 2). Rather, the decrease in locomotor activity was mirrored by a decrease in category 4 behavior (Fig. 5C), rather than accompanied by an increase in stereotypy, as would be indicated if stereotyped behavior interfered with a further stimulation of locomotor activity.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Dose-related alteration by TZP of cocaine effects on extracellular DA in the NAc shell, locomotor activity, and stereotyped behavior. Filled points represent the effects of cocaine alone; open points represent effects of the increasing doses of TZP, administered s.c. 20 min before cocaine. Data are from the first 30 min after cocaine administration during which its effects were maximal. The unconnected filled point above Sal represents the effects during the 30 min after the second of two vehicle injections; open points above Sal represent the effects during the 30 min after a vehicle injection after TZP injection. Vertical bars, where present represent S.E.M., and where absent are encompassed by the symbol. A, microdialysis data from the first 30 min extracted from those shown in Fig. 2, with the addition of data obtained after injection of TZP alone. B, locomotor activity data from the first 30 min extracted from those shown in Fig. 4. C, frequencies of behavioral category 4 from each of the six subjects observed during the first 30 min after injection of cocaine, TZP alone, or their combination. Frequencies are expressed as percentages of occasions in which the behavior was judged as falling into category 4. Note that TZP increased the effects of cocaine on extracellular DA levels in the NAc shell, whereas both behavioral effects were decreased by TZP.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, as reported previously (Kuczenski et al., 1991; Tanda et al., 1997, 2005), cocaine dose-dependently increased DA levels in the NAc shell, NAc core, and the PFCX. Also in agreement with previous studies, systemic treatment with the preferential M₁ antagonists TZP and TXP did not significantly modify DA levels (Ichikawa et al., 2002; Shimosato et al., 2003). However, both TZP and TXP dose-dependently increased the effects of cocaine on DA in the NAc shell, whereas these effects were absent in the NAc core and the PFCX. TXP stimulated locomotor activity in rats, although TZP did not. TZP attenuated the locomotor stimulant effects of cocaine, whereas TXP did not. Neither drug potentiated cocaine-induced stereotypy. Finally, the present binding studies, albeit limited, extended the preferential affinity of particularly TZP for M₁ over the other subtypes of muscarinic receptors (Eltze et al., 1985; Doods et al., 1987; Bymaster et al., 1993) and confirmed that a preferential binding profile among subtypes of muscarinic receptors was obtained with several analogs of benztropine.

Previous studies on the role of muscarinic receptors on DA levels have provided conflicting results. Many of these studies were performed using local administration or perfusion (reverse dialysis through the probe) of muscarinic agonists and antagonists (Xu et al., 1989; Ichikawa et al., 2002; Rahman and McBride, 2002), which probably produced effects through populations of receptors more restricted than those affected by systemic injection (Ichikawa et al., 2002; Shimosato et al., 2003). However, consistent with the present study, systemic administration of TXP had no effects on DA levels in the NAc or striatum (Ichikawa et al., 2002; Shimosato et al., 2003).

Previous results from pretreatments with antimuscarinic agents on psychostimulant-induced elevations in DA levels have also varied. Rahman and McBride (2002) found that local NAc administration of scopolamine reduced the effects of the DA uptake inhibitor GBR 12909, whereas Sziraki et al. (1998) reported no effect of a selected dose of i.v. atropine on cocaine-induced DA levels in the NAc. Ichikawa et al. (2002) found some increase in the effects of amphetamine on DA levels in the PFCX but not in the NAc with systemic scopolamine administration. Pretreatment with the preferential M₁ antagonist TXP at 5.0 mg/kg attenuated the stimulation of DA levels in the NAc produced by 1.0 mg/kg methamphetamine but not 10 mg/kg cocaine (Shimosato et al., 2003). In any attempt to reconcile the aforementioned findings, it should be noted that in the present study as little as a 3-fold change in dose changed either antagonist from ineffective to effective in the enhancement of the effects of cocaine on extracellular DA (Figs. 2 and 3).

There was a marked potentiation of the effects of cocaine on DA levels in the NAc shell produced by both M₁ antagonists that was not obtained in the NAc core or the PFCX. These differences indicate a greater contribution of M₁ receptors to DA activity in the NAc shell than in the other areas. Moreover, these regional differences indicate that probe placement may also have to be considered in reconciling conflicting results in the literature.

Systemic administration of M₁ receptor antagonists may, by a feedback loop, increase the release of acetylcholine, thereby activating other muscarinic receptors in addition to M₁ subtypes. Through that mechanism, for example, activation of M₅ receptors located in the VTA could facilitate the firing of VTA DA neurons (Fink-Jensen et al., 2003), which are typically more sensitive than substantia nigra DA neurons to the effects of many different classes of drugs (Gessa et al., 1985; Mereu et al., 1987). Further studies of this mechanism might explain the differences in the effects of M₁ antagonists on cocaine-induced increases in DA levels in the shell (mostly innervated by the VTA) compared with the core of the NAc (Zahm, 1999).

Previous behavioral studies of the effects of combinations of cocaine and antimuscarinics have focused primarily on the nonselective antagonists atropine and scopolamine. For example, both drugs augment stimulant effects of amphetamine (Carlton, 1961) and cocaine (Scheckel and Boff, 1964; Wilson and Schuster, 1973; Katz et al., 1999). In addition, studies in muscarinic M₁ receptor knockout mice indicate that a deficiency of M₁ receptors produces increases in dopaminergic tone, increases in locomotor activity, and an increased responsiveness to the stimulant effects of amphetamine (Gerber et al., 2001). Thus, the past results taken together suggest that interfering with M₁ muscarinic receptor action increases sensitivity to psychomotor stimulant drugs.

The present results have shown that TXP administration alone increased locomotor activity in rats, and that in combination with cocaine, TXP enhanced the locomotor stimulant effects of cocaine. Atropine and scopolamine have been shown to enhance the locomotor stimulant effects of cocaine, and a similar result with TXP was reported previously (Shimosato et al., 2001). However, a lack of effect of TXP on a cocaine place conditioning was also reported (Shimosato et al., 2001). In contrast, TZP attenuated the effects of cocaine on locomotor activity. Thus, the present results suggest a more complicated picture for the influence of muscarinic antagonism on this behavioral effect of cocaine, or additional actions of at least one of the antagonists.

Both TXP and TZP increased the effects of cocaine on DA levels in the NAc shell, whereas their effects on cocaine-induced locomotor activity were different, with only TZP attenuating the effects of cocaine. The locomotor stimulant effects of cocaine are thought to be derived from its ability to increase DA neurotransmission particularly in limbic areas (Ikemoto, 2002). As a consequence, it should be expected, as it occurred with TXP, that the selective increase of DA levels in the NAc shell obtained when animals were pretreated with either M₁ antagonist would facilitate this behavioral effect of cocaine.

The different interactions of TXP and TZP with cocaine on locomotor activity might be reconciled if TZP with cocaine produced a greater induction of stereotyped behavior than did TXP with cocaine. A sufficiently large effect on stereotypy might be expected to interfere with, and in turn, decrease locomotor activity. Therefore, we tested the hypothesis that the M₁ antagonists potentiated the effects of cocaine, thereby increasing stereotypy. However, had either M₁ antagonist potentiated a cocaine-induced stereotypy, there would have been an enhancement of frequencies of category 4 behaviors or the appearance of categories of behavior with designations greater than 4. Because neither of these outcomes was obtained (Fig. 5C; Table 2), it seems that the two M₁ antagonists had different interactions with cocaine on locomotor activity and that those effects were not simply resulting from a greater induction of interfering stereotypy with TZP compared with TXP.

The differences among neurochemical and behavioral results suggest that the increases in DA levels in the NAc shell do not entirely account for the behavioral effects. It is possible that M₁ antagonism reduces the behavioral effects of cocaine through some mechanism other than through effects on DA levels. However, that both M₁ antagonists did not uniformly attenuate all of the behavioral effects of cocaine suggests otherwise. It is also possible that some unknown action of either of the drugs influenced outcome. Several previous studies focused on DA transporter actions of TXP (Shimosato et al., 2001; Dar et al., 2005). The present stimulation of locomotor activity when TXP was administered alone is consistent with an effect at that site. However, the DA transporter affinity of TXP, although higher than that for TZP, was approximately 4 orders of magnitude lower than its estimated affinity for M₁ sites (also see Dar et al., 2005). In addition, TXP alone did not alter levels of extracellular DA in any of the regions studied, as would be expected with DA transport inhibition. These latter considerations suggest that activity at the DA transporter was an unlikely contributor to the observed effects of TXP.

Stimulation of DA transmission in the NAc shell has been implicated in many behavioral processes, including reinforcing and addictive effects of drugs abused by humans. The present results clearly show that a preferential blockade of muscarinic M₁ receptors did not reduce the effects of cocaine on DA transmission in any of the areas studied but rather that both drugs selectively enhanced the effects of cocaine in the NAc shell, compared with the core or the PFCX. Those results suggest that blockade of M₁ receptors might increase rather than decrease the reinforcing effects of cocaine. We were especially interested in the interactions of TZP and TXP with cocaine, because these compounds have preferential affinities for M₁ over other muscarinic receptors approximating those of the BZT analogs. As described above, if the antimuscarinic activity of BZT analogs interferes with their cocaine-like effects, a muscarinic antagonist should reduce the effects of cocaine. Previous studies found little support for the hypothesis (Katz et al., 1999; Li et al., 2005), although they were conducted with nonselective muscarinic antagonists. The present study further suggests little, if any, support for the hypothesis that M₁ muscarinic receptor antagonism could be a factor contributing to a reduction in the cocaine-like behavioral effects of BZT analogs.

	Acknowledgements

 
We thank Patty Ballerstadt for administrative assistance.

	Footnotes

 
This study was supported by the Intramural Research Program, Department of Health and Human Services, National Institute on Drug Abuse, National Institutes of Health.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.118067.

ABBREVIATIONS: DA, dopamine; NAc, nucleus accumbens; BZT, benztropine; TZP, telenzepine; TXP, trihexyphenidyl; WIN 35,428, 2-carbomethoxy-3-(4-fluorophenyl)tropane; PEI, polyethyleneimine; PFCX, prefrontal cortex; AHN 1-055, [4'-4''-difluoro-3-(diphenylmethoxy)-tropane]; 4'-4''-diCl-BZT, [4'-4''-dichloro-3-(diphenylmethoxy)-tropane]; 4-Cl-BZT, [4'-chloro-3-(diphenylmethoxy)-tropane]; ANOVA, analysis of variance; GBR 12909, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; VTA, ventral tegmental area.

Address correspondence to: Dr. Gianluigi Tanda, Department of Health and Human Services, National Institute on Drug Abuse, National Institutes of Health, Intramural Research Program, Medications Discovery Research Branch, Psychobiology Section, 5500 Nathan Shock Dr., Baltimore, MD 21224. E-mail: gtanda{at}intra.nida.nih.gov

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