Evidence for the involvement of dopamine transporters in behavioral stimulant effects of modafinil

Abstract

Modafinil is prescribed for numerous medical conditions, but the drug's mechanism of action is unclear. Here, we examined the interaction of modafinil with receptors and transporters in vitro and compared pharmacological effects of the drug with those produced by indirect dopamine (DA) agonists 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) and (+)-methamphetamine (METH). Modafinil was screened at various receptors and transporters using binding assays. Transporter-mediated uptake and release were examined in rat brain synaptosomes. Effects of modafinil on motor activity and neurochemistry were determined in rats undergoing in vivo microdialysis in nucleus accumbens. Of the receptors and transporters assayed, modafinil displayed measurable potency only at DA transporters (DAT), inhibiting [(3)H]DA uptake, with an IC(50) value of 4.0 microM. Accordingly, modafinil pretreatment (10 microM) antagonized METH-induced release of the DAT substrate [(3)H]1-methyl-4-phenylpyridinium. Intravenous modafinil (20 and 60 mg/kg) produced dose-dependent increases in motor activity and extracellular DA, without affecting serotonin (5-HT). Analogous results were observed for GBR12909 (1 and 3 mg/kg), whereas METH (0.3 and 1 mg/kg) increased DA and 5-HT. Locomotor effects of all drugs were positively correlated with dialysate DA (P < 0.001). Interestingly, modafinil pretreatment reduced METH-induced ambulation and DA release. Our data show that modafinil interacts with DAT sites in rat brain, a property shared with agonist medications under investigation for treating cocaine dependence. Nondopaminergic mechanisms may also contribute to the pharmacology of modafinil. Finally, the results suggest that modafinil should be tested as an adjunct for treating METH addiction.

Fig. 1.

Effects of modafinil on inhibition of [³H]amine uptake. Various concentrations of modafinil were incubated with [³H]DA or [³H]NE in rat brain synaptosomes as described under Materials and Methods. Values are mean ± S.D. expressed as percentage of control uptake determined from three separate experiments.

Fig. 2.

Effects of modafinil on METH-induced release of [³H]MPP⁺ (top) and [³H]5-HT (bottom). Various concentrations of METH were added to synaptosomes preloaded with radiolabeled substrate for DAT ([³H]MPP⁺) or SERT ([³H]5-HT). Release assays were conducted in the presence or absence of 10 μM modafinil. Values are mean ± S.D. expressed as percentage of control tritium retained determined from three separate experiments.

Fig. 3.

Effects of modafinil on ambulation (top) and stereotypy (bottom) in rats undergoing microdialysis in the nucleus accumbens. Rats received intravenous modafinil or vehicle at time 0. Ambulation and stereotypy were measured in 20-min bins. Ambulation is expressed as distance traveled in centimeters, whereas stereotypy is expressed as number of moves. Values are mean ± S.E.M. for n = 6 rats/group. Asterisks represent significant effects compared with vehicle control at particular time points (Newman-Keuls, P < 0.05).

Fig. 4.

Effects of modafinil on extracellular DA (top) and 5-HT (bottom) in rats undergoing microdialysis in the n. accumbens. Rats received intravenous modafinil or vehicle at time 0. Dialysate samples were collected at 20-min intervals and assayed for DA and 5-HT via HPLC-ECD. Values are mean ± S.E.M. expressed as picograms/5-μl sample for n = 6 rats/group. Asterisks represent significant effects compared with vehicle control at particular time points (Newman-Keuls, P < 0.05).

Fig. 5.

Effects of the DA uptake inhibitor GBR12909 on ambulation (top) and stereotypy (bottom) in rats undergoing microdialysis in the n. accumbens. Rats received intravenous GBR12909 or saline at time 0. Ambulation and stereotypy were measured in 20-min bins. Ambulation is expressed as distance traveled in centimeters, whereas stereotypy is expressed as number of moves. Values are mean ± S.E.M. for n = 6 rats/group. Asterisks represent significant effects compared with saline control at particular time points (Newman-Keuls, P < 0.05).

Fig. 6.

Effects of GBR12909 on extracellular DA (top) and 5-HT (bottom) in rats undergoing microdialysis in the n. accumbens. Rats received intravenous GBR12909 or saline at time 0. Dialysate samples were collected at 20-min intervals and assayed for DA and 5-HT via HPLC-ECD. Values are mean ± S.E.M. expressed as picograms/5-μl sample for n = 6 rats/group. Asterisks represent significant effects compared with saline control at particular time points (Newman-Keuls, P < 0.05).

Fig. 7.

Effects of the releasing agent METH on ambulation (top) and stereotypy (bottom) in rats undergoing microdialysis in the n. accumbens. Rats received i.v. METH or saline at time 0. Ambulation and stereotypy were measured in 20-min bins. Ambulation is expressed as distance traveled in centimeters, whereas stereotypy is expressed as number of moves. Values are mean ± S.E.M. for n = 6 rats/group. Asterisks represent significant effects compared with saline control at particular time points (Newman-Keuls, P < 0.05).

Fig. 8.

Effects of METH on extracellular DA (top) and 5-HT (bottom) in rats undergoing microdialysis in the n. accumbens. Rats received intravenous METH or saline at time 0. Dialysate samples were collected at 20-min intervals and assayed for DA and 5-HT via HPLC-ECD. Values are mean ± S.E.M. expressed as picograms/5-μl sample for n = 6 rats/group. Asterisks represent significant effects compared with saline control at particular time points (Newman-Keuls, P < 0.05).

Fig. 9.

Correlations between motor stimulation and dialysate DA responses produced by intravenous modafinil. Raw data from the first 60 min after injection of modafinil (20 and 60 mg/kg i.v.) and vehicle were used to construct correlation plots (see Figs. 3 and 4). Fifty-four data points contributed to correlations for ambulation (centimeters) versus DA (picograms) and stereotypy (number of moves) versus DA (picograms). Pearson correlation coefficients (r), slopes of the best-fit linear regression (m), and P values for statistical significance are given.

Fig. 10.

Correlations between motor stimulation and dialysate DA responses produced by intravenous GBR12909. Raw data from the first 60 min after injection of GBR12909 (1 and 3 mg/kg i.v.) and saline were used to construct correlation plots (see Figs. 5 and 6). Fifty-four data points contributed to correlations for ambulation (centimeters) versus DA (picograms) and stereotypy (number of moves) versus DA (picograms). Pearson correlation coefficients (r), slopes of the best-fit linear regression (m), and P values for statistical significance are given.

Fig. 11.

Correlations between motor stimulation and dialysate DA responses produced by intravenous METH. Raw data from the first 60 min after injection of METH (0.3 and 1.0 mg/kg i.v.) and saline were used to construct correlation plots (see Figs. 7 and 8). Fifty-four data points contributed to correlations for ambulation (centimeters) versus DA (picograms) and stereotypy (number of moves) versus DA (picograms). Pearson correlation coefficients (r), slopes of the best-fit linear regression (m), and P values for statistical significance are given.

Fig. 12.

Effects of modafinil pretreatment on ambulation produced by intravenous saline (top) or METH (bottom) in rats undergoing microdialysis in n. accumbens. Pretreatment with intravenous vehicle or modafinil (20 mg/kg) was administered at time 0, followed by intravenous saline or METH (0.3 mg/kg) given 40 min later. Ambulation was measured in 20-min bins. Data are mean ± S.E.M. expressed as centimeters traveled for n = 8 rats/group. Asterisks represent significance with respect to vehicle plus METH treatment group at particular time points (Newman-Keuls, P < 0.05).

Fig. 13.

Effects of modafinil pretreatment on stereotypy produced by intravenous saline (top) or METH (bottom) in rats undergoing microdialysis in n. accumbens. Pretreatment with intravenous vehicle or modafinil (20 mg/kg) was administered at time 0, followed by intravenous saline or METH (0.3 mg/kg) given 40 min later. Stereotypy was measured in 20-min bins. Data are mean ± S.E.M. expressed as number of moves for n = 8 rats/group.

Fig. 14.

Effects of modafinil pretreatment on the dialysate DA response produced by intravenous saline (top) or METH (bottom) in rats undergoing microdialysis in the n. accumbens. Pretreatment with intravenous vehicle or modafinil (20 mg/kg) was administered at time 0, followed by intravenous saline or METH (0.3 mg/kg) given 40 min later. Dialysate samples were collected every 20 min and assayed for DA by HPLC-ECD. Data are mean ± S.E.M. expressed as picograms/5-μl sample for n = 8 rats/group. Asterisks represent significance with respect to vehicle plus METH treatment group at particular time points (Newman-Keuls, P < 0.05).