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RTI-76, an irreversible inhibitor of dopamine transporter binding, increases locomotor activity in the rat at high doses
Brain Research 897 (2001) 157–163 www.elsevier.com / locate / bres
Research report
RTI-76, an irreversible inhibitor of dopamine transporter binding, increases locomotor activity in the rat at high doses a, b a Heather L. Kimmel *, F. Ivy Carroll , Michael J. Kuhar a
Yerkes Regional Primate Research Center, Emory University, 954 Gatewood Rd. NE, Atlanta, GA 30329, USA b Research Triangle Institute, Research Triangle Park, NC 27709, USA Accepted 18 January 2001
Abstract An earlier study in our laboratory showed that 24 h after intracerebroventricular administration of the irreversible dopamine transporter inhibitor RTI-76, [ 3 H]GBR12935 binding to the dopamine transporter (DAT) protein was inhibited in both the striatum and nucleus accumbens of the rat in a dose-dependent fashion (0.05–5.0 mmol). The rate of return of binding to control levels was used to calculate the half-life of DAT. Since changes in behavior could conceivably influence the half-life, the effects of various doses of RTI-76 on locomotor activity 1 and 3 days after RTI-76 administration were examined. During the first day after i.c.v. administration, 1.25 mmol RTI-76 had no effect on locomotor activity, but 2.5 mmol RTI-76 significantly increased locomotor activity in rats, a time at which this dose inhibited 41 and 42% of [ 3 H]GBR12935 binding in the striatum and in the nucleus accumbens, respectively. These results agree with earlier reports showing that significant blockade of the dopamine transporter protein in the striatum is required for increases in motor activity in rodents. However, 5.0 mmol RTI-76 did not increase locomotor activity, even though binding was inhibited to 38 and 37% of control levels in the striatum and nucleus accumbens, respectively. Furthermore, our present results suggest that locomotor activity does not continue to increase as the blockade of DAT increases. Notably, there were no increases in locomotor activity at the dose of RTI-76 (100 nmol) used to measure DAT half-life. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Uptake and transporters Keywords: Binding; Dopamine transporter; GBR12935; Locomotor activity; Rat; RTI-76
1. Introduction The locomotor activity and reinforcing effects of psychomotor stimulant drugs, such as amphetamine and cocaine, have been attributed, at least in part, to their ability to increase dopamine levels in the brain [3,13,21,22]. Many compounds that increase dopamine levels by inhibiting the dopamine transporter also increase locomotor activity in rodents. For example, these compounds include cocaine and its chemical analogs [2,6,9,20,23,26,27]. The role of DAT in locomotor activity is further emphasized in dopamine transporter knockout
*Corresponding author. Tel.: 11-404-727-1737; fax: 11-404-7273278. E-mail address: [email protected] (H.L. Kimmel).
mice, which exhibit a higher level of baseline locomotor activity [4,8,10]. RTI-76 (3b-( p-chlorophenyl) tropan-2b-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride) is a potent and irreversible ligand for the DAT protein [1]. The irreversible nature of this compound has allowed us to study turnover kinetics of DAT and serotonin transporter (SERT) proteins in the rat by measuring the rate of transporter protein recovery after i.c.v. administration of RTI-76 [12,24]. These studies found that complete recovery of binding to DAT and SERT occurs 7–14 days after administration of RTI-76. Due to its irreversible biochemical effects at the DAT protein, this compound could cause long-lasting increases in locomotor activity, provided a statistically significant fraction of DATs was inhibited. In turn, the increased locomotor activity could influence CNS neuronal activity
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02118-7
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and DAT half-life. Therefore, we measured locomotor activity continuously for 3 days following i.c.v. administration of various doses of RTI-76 in order to assess the behavioral effects of this compound. In a second group of animals, we measured DAT binding in the rat striatum and nucleus accumbens one and 3 days following i.c.v. administration of RTI-76 in order to correlate the inhibition of DAT binding with the observed behavior.
2. Materials and methods
2.1. Behavioral studies 2.1.1. Implantation of chronically indwelling cannulae Male Sprague–Dawley rats (250–350 g, Charles River) were anesthetized with 100 mg / kg pentobarbitol (i.p.) and placed into stereotaxic frame (Kopf Instruments, Tujunga, CA) with the incisor bar set 3.9 mm below the horizontal plane. A cannula was placed just above the right lateral ventricle at the stereotaxic coordinates AP520.8, ML52 1.4, DV524.0, relative to bregma. The cannula was secured to the skull by means of skull screws and dental cement. Animals were allowed to recover for 5–7 days. Placement of the cannulae was verified by measuring polydypsia following the administration of 0.1 mg / 5.0 ml angiotensin II via the cannula. If the animal did not exhibit drinking behavior within 2–3 min after angiotensin administration, they were excluded from the behavioral study. 2.1.2. Locomotor activity Locomotor activity was measured using eight Digiscan animal activity monitors (AccuScan Instruments, Inc., Columbus, OH). On test days, each rat was placed individually inside a polycarbonate cage (16.5316312 inches), which was then placed inside of an activity monitor. Activity was monitored by infrared light beam sensors (eight beams per side) located on two opposing sides of the monitor. Activity was measured with a Digiscan analyzer and then saved to a data file for subsequent analyses. All rats were habituated to the locomotor activity chamber for at least 5 days before testing. 2.1.3. Drug administration Animals received a single administration of 1.25 mmol RTI-76, 2.5 mmol RTI-76, 5.0 mmol RTI-76 or saline injected in 20 ml volume over a 2-min period. The injection needle was left in place in the cannula for 1–2 min following injection to prevent backflow of the solution, and animals were placed into the experimental chamber. Locomotor activity was measured in 10-min intervals continuously for 3 days following drug administration. During the 3-day testing session, animals had free access to food and water inside the testing chamber.
Animals received an injection only once every 7 days to minimize any residual drug effect between drug treatments and testing sessions. By the completion of the study, every animal received each dose of RTI-76 or saline, in a random order. A higher dose of RTI-76 (10 mmol) was administered, but this dose was lethal in every animal tested and discontinued.
2.1.4. Drugs RTI-76 (3b-( p-chlorophenyl) tropan-2b-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride) was prepared as previously reported [1] and dissolved in 0.9% saline. 2.1.5. Behavioral data analysis The total locomotor stimulant effects of RTI-76 were analyzed using a one-factor repeated measures ANOVA. Time course data were collapsed into 100-min bins and analyzed using a two-factor ANOVA with repeated measures on dose and time. Each ANOVA analysis was followed by a Tukey’s post-hoc test for multiple pairwise comparisons. Differences were considered statistically significant if P,0.05. All behavioral data were analyzed using GB-STAT v. 6.5 (Dynamic Microsystems, Silver Spring, MD). 2.2. Radioligand binding assays 2.2.1. Animals A separate group of rats (250–275 g) were housed five per cage with food and water available at all times. Animals were housed in a colony room with a 12-h light / dark cycle. The animals were maintained in accordance with NIH Guide for Care and Use of Laboratory Animals (NIH / 85-23) and all experimental protocols were approved by the Institutional Animal Care and Use Committee. 2.2.2. Surgery Rats were given a unilateral injection of 1.25, 2.5, or 5.0 mmol RTI-76 or saline in the right lateral ventricle. Animals were anesthetized with 400 mg / kg chloral hydrate (i.p.) and placed in a stereotaxic frame. Stereotaxic coordinates used relative to bregma were: AP520.8, ML521.4, DV524.0. A 25-ml Hamilton syringe was used to inject 20 ml of the RTI-76 solution over a 1-min period. Upon completion of the injection, the needle was kept in place for 3 min to minimize the back flow of the solution. Since RTI-76 is a light- and temperature-sensitive compound, all manipulations were done under low-light conditions and the containers were wrapped in foil and kept on ice. 2.2.3. Preparation of membranes Rats were rapidly decapitated at 1 or 3 days following the injection of RTI-76 or saline. Brains were removed
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immediately and placed in cold saline. The olfactory tubercles were removed with the aid of fine forceps, exposing the diagonal bands of Broca. A razor blade was then used to cut away the frontal cortex and to dissect the nucleus accumbens from both hemispheres. The pair of nucleus accumbens was pooled for each animal, for a total weight of 10–20 mg. Fine forceps were used to remove the striatum from both hemispheres, each weighing 20–30 mg. All tissues were removed rapidly and frozen on dry ice, then stored at 2808C until assayed. On the day of the assay, each tissue was weighed and then placed in the appropriate ice-cold assay buffer (see below). The tissue was then homogenized with a Polytron (Brinkman Instruments Co., Cantiague Road, Westbury, NY) (setting 5 for 15 s). The homogenate was centrifuged for 10 min at 30 0003g and the pellet suspended in buffer. The homogenate was centrifuged again and the pellet suspended in buffer.
2.2.4. [ 3 H] GBR12935 binding After the final centrifugation, the tissue pellet was resuspended in buffer for a final concentration of 5 mg wet tissue weight / ml. Polystyrene test tubes were filled with 1.5 ml buffer (50 mM Tris–HCl, pH 7.7 at 248C, containing 120 mM NaCl and 0.01% bovine serum albumin), 100 ml of 5 mm mazindol (final) or buffer, 400 ml of radioligand, and 40 ml of tissue for a total volume of 2.04 ml. Tubes were incubated at room temperature for 60 min, and the incubations were terminated by rapid filtration over Whatman GF / B filters that were pretreated with 0.05% polyethylenimine. The filters were rinsed three times with 4 ml of ice-cold buffer and the radioactivity remaining on the filters was measured by conventional liquid spectrometry. 2.2.5. Chemicals Bovine serum albumin, chloral hydrate, cocaine hydrochloride, mazindol hydrochloride, polyethylenimine, and potassium chloride were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium chloride was obtained from Fisher Scientific (Pittsburgh, PA). Tris hydrochloride and Tris base were obtained from EM Science (Cincinnati, OH). [ 3 H]GBR12935 was obtained from Dupont-New England Nuclear (Boston, MA). RTI-76 (3b-( p-chlorophenyl) tropan-2b-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride) was prepared as previously reported [1] and dissolved in 0.9% saline. Chloral hydrate was dissolved in 0.9% saline and compounds used in the binding assay were dissolved in the appropriate buffer. 2.2.6. Binding data curve fitting Bmax and Kd values were determined in each binding assay by fitting a nonlinear least-squares analysis and by a linear Scatchard plot. Data were analyzed using GB-STAT v. 6.5 (Dynamic Microsystems, Silver Spring, MD). For
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each brain region, individual one-way ANOVAs were used to compare binding to DAT following RTI-76 administration with that following saline administration on day 1 and day 3. A two-way ANOVA was performed on the data for each brain region, comparing the changes in binding from day 1 to day 3 following i.c.v. saline, 1.25 mmol RTI-76, and 2.5 mmol RTI-76. Each ANOVA was followed by a Tukey’s post-hoc test for significant differences between groups.
3. Results Intracerebroventricular administration of 100 nmol RTI76 did not increase locomotor activity in rats (data not shown). The doses of RTI-76 tested were then increased to 1.25–5.0 mmol, and behavior measured in 10-min intervals for 3 days (Fig. 1). These doses of RTI-76 produced a slow onset with peak activity occurring about 20 (2.5 mmol) or 16 h (5.0 mmol) after injection. Activity returned to those of saline levels after 30–50 h. The locomotor activity data exhibited in Fig. 1A–C was collapsed into 100-min bins and analyzed using a two-way ANOVA with repeated measures on dose and time (Fig. 1D). There was no significant overall effect of dose (F(3,21)52.52, NS), but there was a significant effect of time (F(43,901)53.01, P,0.0001) and a significant dose3 time interaction (F(129,903)51.40, P50.0041). A Tukey’s post-hoc test revealed that from 20 to 28.3 h, 2.5 mmol RTI-76 increased locomotor activity significantly greater than saline did. No other doses significantly increased locomotor activity greater than saline did at any other time point analyzed. An examination of the total horizontal activity over the 3-day testing period revealed an effect of RTI-76 (Fig. 2A). Although no individual dose of RTI-76 produced significantly greater locomotor activity than saline did over the 3-day observation period, there was an overall significant effect of RTI-76 treatment (F(3,21)53.27, P50.041). To facilitate the comparison between the behavioral data and the binding data at specific time points, the locomotor activity observed during the final 30 min of days 1 and 3 were collapsed (Fig. 2B). A two-way repeated measures ANOVA revealed a significant effect of day (F(1,12)5 8.42, P50.013) and of RTI-76 dose (F(3,36)54.09, P5 0.0135), but not of day3dose (F(3,36)50.8686, NS). These two time points were selected because, in the binding studies, we sacrificed the animals within 30 min of the time point analyzed. Administration of RTI-76 (1.25–5.0 mmol) dose-dependently and significantly decreased [ 3 H]GBR12935 binding to DAT in the striatum 1 day after i.c.v. administration (F(3,9)541.43, P,0.001) (Fig. 3A). [ 3 H]GBR12935 binding of DAT in the nucleus accumbens was also significantly decreased after i.c.v. administration of RTI-76 (F(3,9)550.10, P,0.001) (Fig. 3B). All three doses of
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Fig. 1. Horizontal locomotor activity measured in 10-min intervals for 3 days after i.c.v. injection of (A) 1.25 mmol, (B) 2.5 mmol, and (C) 5.0 mmol RTI-76, compared with locomotor activity after i.c.v. administration of saline. (D) Time course of horizontal locomotor activity after i.c.v. administration of saline or 1.25–5.0 mmol RTI-76 in 100-min intervals (data replicated from panels A–C). * Significantly different from saline-induced locomotor activity at that time point, P,0.05. For all four panels, each data point represents the average activity of seven animals for that time interval. Dotted lines represent 24-h time intervals. Error bars have been omitted from these four panels for clarity.
RTI-76 significantly decreased binding, with the highest dose decreasing [ 3 H]GBR12935 binding to 37% of control levels in both the striatum and nucleus accumbens. As previously found [12], the Kd values were not significantly altered after the administration of any dose of RTI-76 at these times (not shown). Three days after i.c.v. injection with RTI-76, an increase in [ 3 H]GBR12935 binding to DAT in both brain regions was observed relative to day 1 (Fig. 3). No animals that received 5.0 mmol RTI-76 under anesthesia survived for longer than 1 day after injection, thus only data for 1.25 and 2.5 mmol-treated animals are shown at 3 days. In the striatum, binding observed on day 3 after 1.25 and 2.5 mmol RTI-76 was not significantly different from that observed 3 days following saline administration (F(2,5)5 3.43, NS) (Fig. 3A). A two-way ANOVA revealed that there was a significant effect of time after drug administration (F(1,13)518.25, P50.0009), a significant effect of RTI-76 dose (F(2,13)529.94, P,0.0001), and a significant interaction between these two factors (F(2,13)56.19, P50.01). However, post-hoc tests showed that binding on day 3 was significantly greater than that on day 1 only after i.c.v. administration of 2.5 mmol RTI-76, but not after i.c.v. administration of 1.25 mmol RTI-76.
In the nucleus accumbens (Fig. 3B), there was a significant effect of RTI-76 dose upon binding observed on day 3 after drug administration (F(2,4)531.95, P5 0.0035). At this time point, 1.25 and 2.5 mmol RTI-76 continued to inhibit binding to DAT in a significant manner. A two-way ANOVA revealed that there was a significant effect of time after drug administration (F(1,12)57.24, P50.0196) and a significant effect of RTI76 dose (F(2,12)577.80, P,0.0001), but no significant interaction between these two factors (F(2,12)51.94, NS). While the binding increased from day 1 to day 3, post-hoc tests showed that the binding to DAT in the nucleus accumbens following i.c.v. administration of 1.25 or 2.5 mmol RTI-76 on day 1 was not significantly different from binding on day 3.
4. Discussion As previously reported, intraventricular injection of RTI-76 significantly decreased [ 3 H]GBR12935 binding at the dopamine transporter in the striatum and nucleus accumbens of the rat [12]. Binding of RTI-76 to DAT is irreversible [1] and blocks other DAT ligands from binding
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Fig. 2. (A) Total horizontal locomotor activity measured for 3 days after i.c.v. administration of saline or RTI-76 (1.25–5.0 mmol). There was an overall significant effect of RTI-76 treatment (F(3,21)53.27, P50.041). (B) Total horizontal locomotor activity measured for the last 30 min of days 1 and 3. For both panels, each bar represents the mean and S.E.M. of seven animals.
to these proteins [7,12]. As new DAT proteins are generated and made available to the presynaptic membrane, [ 3 H]GBR12935 binding to DAT increases, returning to baseline levels about 7 days after RTI-76 administration [12]. Thus, RTI-76 is a useful tool for measuring the turnover kinetics of the DAT protein. In the present study, the dose of RTI-76 that produced behavioral changes was determined and the dose correlated to DAT occupancy and locomotor activity. In the present radioligand binding studies, increasing doses of RTI-76 produced dose-related decreases in DAT binding, reaching a plateau of 37% of control binding. In rDAT-LLC-PK1 cells, however, RTI-76 inhibited 95% or more of [ 125 I]RTI-55 binding to DAT [19], suggesting that RTI-76 can reach and bind to all available DAT proteins in cell culture. The remaining transporters that are available for binding in the striatal homogenates may represent a pool of proteins that are not exposed to RTI-76 in in vivo experiments. These additional transporters may be those
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that were more distant from the lateral ventricles and were not exposed to RTI-76. DAT immunogold labeling studies of the rat nucleus accumbens indicated that most DAT proteins were located on synaptic plasma membranes, but that some were associated with intracellular membranes [18]. Intracerebroventricular injection of RTI-76 (100 nmol, 1.25 mmol, 2.5 mmol and 5.0 mmol) in awake animals produced dose-dependent increases in locomotor activity, suggesting that RTI-76 increases extracellular dopamine levels in the mesolimbic dopamine system much like the psychostimulants amphetamine and cocaine. However, 100 nmol, and 1.25 mmol RTI-76 had no effect on locomotor activity. Thus, there was no increase in locomotor activity with the dose (100 nmol) used to measure DAT half-life [12]. Thus changes in locomotor behavior would not influence these measurements of DAT kinetics. The onset of activity was extremely slow, suggesting that this compound either diffused through the brain slowly or did not bind to the transporter rapidly. The time that peak activity was observed (16–20 h) (Fig. 2) corresponded well with the time that maximal inhibition of DAT binding was observed (24 h) [12]. The long duration of action of RTI-76 reflected the irreversibility of this compound. In addition, the time course of recovery of DAT binding in the striatum paralleled the time course of behavior. These data suggest that inhibition of DAT in the striatum may play a larger role in increasing locomotor activity following RTI-76 administration than does inhibition of DAT in the nucleus accumbens. In these experiments, locomotor activity returned to basal levels within 3 days, which corresponded to 75% of control binding in the striatum and 58% in the nucleus accumbens, suggesting that greater than 25–42% blockade of DAT is needed for significant increases in activity. Previous studies indicate that cocaine analogs produce peak locomotor effects when synaptic dopamine transporter sites are occupied by more than 50% [2,16,17]. Similar results have been found in humans [25]. The locomotor activating effects of psychomotor stimulant drugs such as cocaine and amphetamine have been attributed to the subsequent increases in extrasynaptic dopamine levels in the striatum and in the nucleus accumbens following drug administration [3,15]. However, several studies suggest that increases in dopamine levels in the nucleus accumbens, rather than the striatum, are primarily responsible for the observed increases in motor behavior [11,14]. In the present study, locomotor activity returned to baseline levels at the end of the second day after RTI-76 administration, even though a significant blockade of DAT in the nucleus accumbens was observed 3 days after RTI-76 administration. A 2.5-mmol aliquot of RTI-76 increased locomotor activity during the first day, when about 60% of DAT is inhibited in the nucleus accumbens, but we no longer see this increase on the third day, when approximately 40% of DAT is blocked in this brain region
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Fig. 3. [ 3 H]GBR12935 binding to DAT in the striatum (A) and nucleus accumbens (B) after 1 and 3 days after i.c.v. administration of RTI-76. All binding values are represented as a percent of the Bmax observed after i.c.v. saline. ** Significantly different from saline on that day, P,0.01. 1 significantly different from binding observed at that dose on day 1, P,0.05.
(Fig. 3B). These results concur with data suggesting that more than half of the transporters must be occupied to observe behavioral effects [5,16,25]. In summary, we determined that relatively high doses of the irreversible dopamine transporter ligand RTI-76, given i.c.v., increased locomotor activity in the rat. Using radioligand binding assays, we established that these increases in activity were observed only after more than 50% of DAT protein was occupied by RTI-76. These data again suggest that high occupancy of striatal DAT is necessary for this irreversible DAT ligand to increase motor activity in rats. Moreover, the doses used routinely used to measure DAT turnover (100 nmol) had no behavioral effect, suggesting that changes in behavior were not influencing measurement of turnover.
Acknowledgements This research was supported by grants RR00165 (MJK), DA00418 (MJK), DA10732 (MJK), DA005935 (HLK), and DA05477 (FIC). The authors would like to thank Andrew R. Joyce for his expert technical assistance.
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Research report
RTI-76, an irreversible inhibitor of dopamine transporter binding, increases locomotor activity in the rat at high doses a, b a Heather L. Kimmel *, F. Ivy Carroll , Michael J. Kuhar a
Yerkes Regional Primate Research Center, Emory University, 954 Gatewood Rd. NE, Atlanta, GA 30329, USA b Research Triangle Institute, Research Triangle Park, NC 27709, USA Accepted 18 January 2001
Abstract An earlier study in our laboratory showed that 24 h after intracerebroventricular administration of the irreversible dopamine transporter inhibitor RTI-76, [ 3 H]GBR12935 binding to the dopamine transporter (DAT) protein was inhibited in both the striatum and nucleus accumbens of the rat in a dose-dependent fashion (0.05–5.0 mmol). The rate of return of binding to control levels was used to calculate the half-life of DAT. Since changes in behavior could conceivably influence the half-life, the effects of various doses of RTI-76 on locomotor activity 1 and 3 days after RTI-76 administration were examined. During the first day after i.c.v. administration, 1.25 mmol RTI-76 had no effect on locomotor activity, but 2.5 mmol RTI-76 significantly increased locomotor activity in rats, a time at which this dose inhibited 41 and 42% of [ 3 H]GBR12935 binding in the striatum and in the nucleus accumbens, respectively. These results agree with earlier reports showing that significant blockade of the dopamine transporter protein in the striatum is required for increases in motor activity in rodents. However, 5.0 mmol RTI-76 did not increase locomotor activity, even though binding was inhibited to 38 and 37% of control levels in the striatum and nucleus accumbens, respectively. Furthermore, our present results suggest that locomotor activity does not continue to increase as the blockade of DAT increases. Notably, there were no increases in locomotor activity at the dose of RTI-76 (100 nmol) used to measure DAT half-life. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Uptake and transporters Keywords: Binding; Dopamine transporter; GBR12935; Locomotor activity; Rat; RTI-76
1. Introduction The locomotor activity and reinforcing effects of psychomotor stimulant drugs, such as amphetamine and cocaine, have been attributed, at least in part, to their ability to increase dopamine levels in the brain [3,13,21,22]. Many compounds that increase dopamine levels by inhibiting the dopamine transporter also increase locomotor activity in rodents. For example, these compounds include cocaine and its chemical analogs [2,6,9,20,23,26,27]. The role of DAT in locomotor activity is further emphasized in dopamine transporter knockout
*Corresponding author. Tel.: 11-404-727-1737; fax: 11-404-7273278. E-mail address: [email protected] (H.L. Kimmel).
mice, which exhibit a higher level of baseline locomotor activity [4,8,10]. RTI-76 (3b-( p-chlorophenyl) tropan-2b-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride) is a potent and irreversible ligand for the DAT protein [1]. The irreversible nature of this compound has allowed us to study turnover kinetics of DAT and serotonin transporter (SERT) proteins in the rat by measuring the rate of transporter protein recovery after i.c.v. administration of RTI-76 [12,24]. These studies found that complete recovery of binding to DAT and SERT occurs 7–14 days after administration of RTI-76. Due to its irreversible biochemical effects at the DAT protein, this compound could cause long-lasting increases in locomotor activity, provided a statistically significant fraction of DATs was inhibited. In turn, the increased locomotor activity could influence CNS neuronal activity
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02118-7
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and DAT half-life. Therefore, we measured locomotor activity continuously for 3 days following i.c.v. administration of various doses of RTI-76 in order to assess the behavioral effects of this compound. In a second group of animals, we measured DAT binding in the rat striatum and nucleus accumbens one and 3 days following i.c.v. administration of RTI-76 in order to correlate the inhibition of DAT binding with the observed behavior.
2. Materials and methods
2.1. Behavioral studies 2.1.1. Implantation of chronically indwelling cannulae Male Sprague–Dawley rats (250–350 g, Charles River) were anesthetized with 100 mg / kg pentobarbitol (i.p.) and placed into stereotaxic frame (Kopf Instruments, Tujunga, CA) with the incisor bar set 3.9 mm below the horizontal plane. A cannula was placed just above the right lateral ventricle at the stereotaxic coordinates AP520.8, ML52 1.4, DV524.0, relative to bregma. The cannula was secured to the skull by means of skull screws and dental cement. Animals were allowed to recover for 5–7 days. Placement of the cannulae was verified by measuring polydypsia following the administration of 0.1 mg / 5.0 ml angiotensin II via the cannula. If the animal did not exhibit drinking behavior within 2–3 min after angiotensin administration, they were excluded from the behavioral study. 2.1.2. Locomotor activity Locomotor activity was measured using eight Digiscan animal activity monitors (AccuScan Instruments, Inc., Columbus, OH). On test days, each rat was placed individually inside a polycarbonate cage (16.5316312 inches), which was then placed inside of an activity monitor. Activity was monitored by infrared light beam sensors (eight beams per side) located on two opposing sides of the monitor. Activity was measured with a Digiscan analyzer and then saved to a data file for subsequent analyses. All rats were habituated to the locomotor activity chamber for at least 5 days before testing. 2.1.3. Drug administration Animals received a single administration of 1.25 mmol RTI-76, 2.5 mmol RTI-76, 5.0 mmol RTI-76 or saline injected in 20 ml volume over a 2-min period. The injection needle was left in place in the cannula for 1–2 min following injection to prevent backflow of the solution, and animals were placed into the experimental chamber. Locomotor activity was measured in 10-min intervals continuously for 3 days following drug administration. During the 3-day testing session, animals had free access to food and water inside the testing chamber.
Animals received an injection only once every 7 days to minimize any residual drug effect between drug treatments and testing sessions. By the completion of the study, every animal received each dose of RTI-76 or saline, in a random order. A higher dose of RTI-76 (10 mmol) was administered, but this dose was lethal in every animal tested and discontinued.
2.1.4. Drugs RTI-76 (3b-( p-chlorophenyl) tropan-2b-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride) was prepared as previously reported [1] and dissolved in 0.9% saline. 2.1.5. Behavioral data analysis The total locomotor stimulant effects of RTI-76 were analyzed using a one-factor repeated measures ANOVA. Time course data were collapsed into 100-min bins and analyzed using a two-factor ANOVA with repeated measures on dose and time. Each ANOVA analysis was followed by a Tukey’s post-hoc test for multiple pairwise comparisons. Differences were considered statistically significant if P,0.05. All behavioral data were analyzed using GB-STAT v. 6.5 (Dynamic Microsystems, Silver Spring, MD). 2.2. Radioligand binding assays 2.2.1. Animals A separate group of rats (250–275 g) were housed five per cage with food and water available at all times. Animals were housed in a colony room with a 12-h light / dark cycle. The animals were maintained in accordance with NIH Guide for Care and Use of Laboratory Animals (NIH / 85-23) and all experimental protocols were approved by the Institutional Animal Care and Use Committee. 2.2.2. Surgery Rats were given a unilateral injection of 1.25, 2.5, or 5.0 mmol RTI-76 or saline in the right lateral ventricle. Animals were anesthetized with 400 mg / kg chloral hydrate (i.p.) and placed in a stereotaxic frame. Stereotaxic coordinates used relative to bregma were: AP520.8, ML521.4, DV524.0. A 25-ml Hamilton syringe was used to inject 20 ml of the RTI-76 solution over a 1-min period. Upon completion of the injection, the needle was kept in place for 3 min to minimize the back flow of the solution. Since RTI-76 is a light- and temperature-sensitive compound, all manipulations were done under low-light conditions and the containers were wrapped in foil and kept on ice. 2.2.3. Preparation of membranes Rats were rapidly decapitated at 1 or 3 days following the injection of RTI-76 or saline. Brains were removed
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immediately and placed in cold saline. The olfactory tubercles were removed with the aid of fine forceps, exposing the diagonal bands of Broca. A razor blade was then used to cut away the frontal cortex and to dissect the nucleus accumbens from both hemispheres. The pair of nucleus accumbens was pooled for each animal, for a total weight of 10–20 mg. Fine forceps were used to remove the striatum from both hemispheres, each weighing 20–30 mg. All tissues were removed rapidly and frozen on dry ice, then stored at 2808C until assayed. On the day of the assay, each tissue was weighed and then placed in the appropriate ice-cold assay buffer (see below). The tissue was then homogenized with a Polytron (Brinkman Instruments Co., Cantiague Road, Westbury, NY) (setting 5 for 15 s). The homogenate was centrifuged for 10 min at 30 0003g and the pellet suspended in buffer. The homogenate was centrifuged again and the pellet suspended in buffer.
2.2.4. [ 3 H] GBR12935 binding After the final centrifugation, the tissue pellet was resuspended in buffer for a final concentration of 5 mg wet tissue weight / ml. Polystyrene test tubes were filled with 1.5 ml buffer (50 mM Tris–HCl, pH 7.7 at 248C, containing 120 mM NaCl and 0.01% bovine serum albumin), 100 ml of 5 mm mazindol (final) or buffer, 400 ml of radioligand, and 40 ml of tissue for a total volume of 2.04 ml. Tubes were incubated at room temperature for 60 min, and the incubations were terminated by rapid filtration over Whatman GF / B filters that were pretreated with 0.05% polyethylenimine. The filters were rinsed three times with 4 ml of ice-cold buffer and the radioactivity remaining on the filters was measured by conventional liquid spectrometry. 2.2.5. Chemicals Bovine serum albumin, chloral hydrate, cocaine hydrochloride, mazindol hydrochloride, polyethylenimine, and potassium chloride were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium chloride was obtained from Fisher Scientific (Pittsburgh, PA). Tris hydrochloride and Tris base were obtained from EM Science (Cincinnati, OH). [ 3 H]GBR12935 was obtained from Dupont-New England Nuclear (Boston, MA). RTI-76 (3b-( p-chlorophenyl) tropan-2b-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride) was prepared as previously reported [1] and dissolved in 0.9% saline. Chloral hydrate was dissolved in 0.9% saline and compounds used in the binding assay were dissolved in the appropriate buffer. 2.2.6. Binding data curve fitting Bmax and Kd values were determined in each binding assay by fitting a nonlinear least-squares analysis and by a linear Scatchard plot. Data were analyzed using GB-STAT v. 6.5 (Dynamic Microsystems, Silver Spring, MD). For
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each brain region, individual one-way ANOVAs were used to compare binding to DAT following RTI-76 administration with that following saline administration on day 1 and day 3. A two-way ANOVA was performed on the data for each brain region, comparing the changes in binding from day 1 to day 3 following i.c.v. saline, 1.25 mmol RTI-76, and 2.5 mmol RTI-76. Each ANOVA was followed by a Tukey’s post-hoc test for significant differences between groups.
3. Results Intracerebroventricular administration of 100 nmol RTI76 did not increase locomotor activity in rats (data not shown). The doses of RTI-76 tested were then increased to 1.25–5.0 mmol, and behavior measured in 10-min intervals for 3 days (Fig. 1). These doses of RTI-76 produced a slow onset with peak activity occurring about 20 (2.5 mmol) or 16 h (5.0 mmol) after injection. Activity returned to those of saline levels after 30–50 h. The locomotor activity data exhibited in Fig. 1A–C was collapsed into 100-min bins and analyzed using a two-way ANOVA with repeated measures on dose and time (Fig. 1D). There was no significant overall effect of dose (F(3,21)52.52, NS), but there was a significant effect of time (F(43,901)53.01, P,0.0001) and a significant dose3 time interaction (F(129,903)51.40, P50.0041). A Tukey’s post-hoc test revealed that from 20 to 28.3 h, 2.5 mmol RTI-76 increased locomotor activity significantly greater than saline did. No other doses significantly increased locomotor activity greater than saline did at any other time point analyzed. An examination of the total horizontal activity over the 3-day testing period revealed an effect of RTI-76 (Fig. 2A). Although no individual dose of RTI-76 produced significantly greater locomotor activity than saline did over the 3-day observation period, there was an overall significant effect of RTI-76 treatment (F(3,21)53.27, P50.041). To facilitate the comparison between the behavioral data and the binding data at specific time points, the locomotor activity observed during the final 30 min of days 1 and 3 were collapsed (Fig. 2B). A two-way repeated measures ANOVA revealed a significant effect of day (F(1,12)5 8.42, P50.013) and of RTI-76 dose (F(3,36)54.09, P5 0.0135), but not of day3dose (F(3,36)50.8686, NS). These two time points were selected because, in the binding studies, we sacrificed the animals within 30 min of the time point analyzed. Administration of RTI-76 (1.25–5.0 mmol) dose-dependently and significantly decreased [ 3 H]GBR12935 binding to DAT in the striatum 1 day after i.c.v. administration (F(3,9)541.43, P,0.001) (Fig. 3A). [ 3 H]GBR12935 binding of DAT in the nucleus accumbens was also significantly decreased after i.c.v. administration of RTI-76 (F(3,9)550.10, P,0.001) (Fig. 3B). All three doses of
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Fig. 1. Horizontal locomotor activity measured in 10-min intervals for 3 days after i.c.v. injection of (A) 1.25 mmol, (B) 2.5 mmol, and (C) 5.0 mmol RTI-76, compared with locomotor activity after i.c.v. administration of saline. (D) Time course of horizontal locomotor activity after i.c.v. administration of saline or 1.25–5.0 mmol RTI-76 in 100-min intervals (data replicated from panels A–C). * Significantly different from saline-induced locomotor activity at that time point, P,0.05. For all four panels, each data point represents the average activity of seven animals for that time interval. Dotted lines represent 24-h time intervals. Error bars have been omitted from these four panels for clarity.
RTI-76 significantly decreased binding, with the highest dose decreasing [ 3 H]GBR12935 binding to 37% of control levels in both the striatum and nucleus accumbens. As previously found [12], the Kd values were not significantly altered after the administration of any dose of RTI-76 at these times (not shown). Three days after i.c.v. injection with RTI-76, an increase in [ 3 H]GBR12935 binding to DAT in both brain regions was observed relative to day 1 (Fig. 3). No animals that received 5.0 mmol RTI-76 under anesthesia survived for longer than 1 day after injection, thus only data for 1.25 and 2.5 mmol-treated animals are shown at 3 days. In the striatum, binding observed on day 3 after 1.25 and 2.5 mmol RTI-76 was not significantly different from that observed 3 days following saline administration (F(2,5)5 3.43, NS) (Fig. 3A). A two-way ANOVA revealed that there was a significant effect of time after drug administration (F(1,13)518.25, P50.0009), a significant effect of RTI-76 dose (F(2,13)529.94, P,0.0001), and a significant interaction between these two factors (F(2,13)56.19, P50.01). However, post-hoc tests showed that binding on day 3 was significantly greater than that on day 1 only after i.c.v. administration of 2.5 mmol RTI-76, but not after i.c.v. administration of 1.25 mmol RTI-76.
In the nucleus accumbens (Fig. 3B), there was a significant effect of RTI-76 dose upon binding observed on day 3 after drug administration (F(2,4)531.95, P5 0.0035). At this time point, 1.25 and 2.5 mmol RTI-76 continued to inhibit binding to DAT in a significant manner. A two-way ANOVA revealed that there was a significant effect of time after drug administration (F(1,12)57.24, P50.0196) and a significant effect of RTI76 dose (F(2,12)577.80, P,0.0001), but no significant interaction between these two factors (F(2,12)51.94, NS). While the binding increased from day 1 to day 3, post-hoc tests showed that the binding to DAT in the nucleus accumbens following i.c.v. administration of 1.25 or 2.5 mmol RTI-76 on day 1 was not significantly different from binding on day 3.
4. Discussion As previously reported, intraventricular injection of RTI-76 significantly decreased [ 3 H]GBR12935 binding at the dopamine transporter in the striatum and nucleus accumbens of the rat [12]. Binding of RTI-76 to DAT is irreversible [1] and blocks other DAT ligands from binding
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Fig. 2. (A) Total horizontal locomotor activity measured for 3 days after i.c.v. administration of saline or RTI-76 (1.25–5.0 mmol). There was an overall significant effect of RTI-76 treatment (F(3,21)53.27, P50.041). (B) Total horizontal locomotor activity measured for the last 30 min of days 1 and 3. For both panels, each bar represents the mean and S.E.M. of seven animals.
to these proteins [7,12]. As new DAT proteins are generated and made available to the presynaptic membrane, [ 3 H]GBR12935 binding to DAT increases, returning to baseline levels about 7 days after RTI-76 administration [12]. Thus, RTI-76 is a useful tool for measuring the turnover kinetics of the DAT protein. In the present study, the dose of RTI-76 that produced behavioral changes was determined and the dose correlated to DAT occupancy and locomotor activity. In the present radioligand binding studies, increasing doses of RTI-76 produced dose-related decreases in DAT binding, reaching a plateau of 37% of control binding. In rDAT-LLC-PK1 cells, however, RTI-76 inhibited 95% or more of [ 125 I]RTI-55 binding to DAT [19], suggesting that RTI-76 can reach and bind to all available DAT proteins in cell culture. The remaining transporters that are available for binding in the striatal homogenates may represent a pool of proteins that are not exposed to RTI-76 in in vivo experiments. These additional transporters may be those
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that were more distant from the lateral ventricles and were not exposed to RTI-76. DAT immunogold labeling studies of the rat nucleus accumbens indicated that most DAT proteins were located on synaptic plasma membranes, but that some were associated with intracellular membranes [18]. Intracerebroventricular injection of RTI-76 (100 nmol, 1.25 mmol, 2.5 mmol and 5.0 mmol) in awake animals produced dose-dependent increases in locomotor activity, suggesting that RTI-76 increases extracellular dopamine levels in the mesolimbic dopamine system much like the psychostimulants amphetamine and cocaine. However, 100 nmol, and 1.25 mmol RTI-76 had no effect on locomotor activity. Thus, there was no increase in locomotor activity with the dose (100 nmol) used to measure DAT half-life [12]. Thus changes in locomotor behavior would not influence these measurements of DAT kinetics. The onset of activity was extremely slow, suggesting that this compound either diffused through the brain slowly or did not bind to the transporter rapidly. The time that peak activity was observed (16–20 h) (Fig. 2) corresponded well with the time that maximal inhibition of DAT binding was observed (24 h) [12]. The long duration of action of RTI-76 reflected the irreversibility of this compound. In addition, the time course of recovery of DAT binding in the striatum paralleled the time course of behavior. These data suggest that inhibition of DAT in the striatum may play a larger role in increasing locomotor activity following RTI-76 administration than does inhibition of DAT in the nucleus accumbens. In these experiments, locomotor activity returned to basal levels within 3 days, which corresponded to 75% of control binding in the striatum and 58% in the nucleus accumbens, suggesting that greater than 25–42% blockade of DAT is needed for significant increases in activity. Previous studies indicate that cocaine analogs produce peak locomotor effects when synaptic dopamine transporter sites are occupied by more than 50% [2,16,17]. Similar results have been found in humans [25]. The locomotor activating effects of psychomotor stimulant drugs such as cocaine and amphetamine have been attributed to the subsequent increases in extrasynaptic dopamine levels in the striatum and in the nucleus accumbens following drug administration [3,15]. However, several studies suggest that increases in dopamine levels in the nucleus accumbens, rather than the striatum, are primarily responsible for the observed increases in motor behavior [11,14]. In the present study, locomotor activity returned to baseline levels at the end of the second day after RTI-76 administration, even though a significant blockade of DAT in the nucleus accumbens was observed 3 days after RTI-76 administration. A 2.5-mmol aliquot of RTI-76 increased locomotor activity during the first day, when about 60% of DAT is inhibited in the nucleus accumbens, but we no longer see this increase on the third day, when approximately 40% of DAT is blocked in this brain region
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Fig. 3. [ 3 H]GBR12935 binding to DAT in the striatum (A) and nucleus accumbens (B) after 1 and 3 days after i.c.v. administration of RTI-76. All binding values are represented as a percent of the Bmax observed after i.c.v. saline. ** Significantly different from saline on that day, P,0.01. 1 significantly different from binding observed at that dose on day 1, P,0.05.
(Fig. 3B). These results concur with data suggesting that more than half of the transporters must be occupied to observe behavioral effects [5,16,25]. In summary, we determined that relatively high doses of the irreversible dopamine transporter ligand RTI-76, given i.c.v., increased locomotor activity in the rat. Using radioligand binding assays, we established that these increases in activity were observed only after more than 50% of DAT protein was occupied by RTI-76. These data again suggest that high occupancy of striatal DAT is necessary for this irreversible DAT ligand to increase motor activity in rats. Moreover, the doses used routinely used to measure DAT turnover (100 nmol) had no behavioral effect, suggesting that changes in behavior were not influencing measurement of turnover.
Acknowledgements This research was supported by grants RR00165 (MJK), DA00418 (MJK), DA10732 (MJK), DA005935 (HLK), and DA05477 (FIC). The authors would like to thank Andrew R. Joyce for his expert technical assistance.
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