Adult male Sprague Dawley rats (250~300 g) were obtained from Hyo-Chang Science Co. (Taegu, Korea). Rats were individually housed in a controlled environment during all experimental treatments. Food and water were provided ad libitum and rats were maintained on a 12 h light/dark cycle. On the day of the experiments, injections were given in a home cage in a quiet room to minimize stress to the animals. All animal use procedures were approved by the Institutional Animal Care and Use Committee and performed in accordance with the provisions of the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Rats were anesthetized with intraperitoneal (i.p.) injections of chloral hydrate (6 ml/kg) and placed in a Stoelting stereotaxic apparatus. Under aseptic conditions, a 23-gauge stainless steel guide cannula (0.29 mm inner diameter, 10 mm in length) was implanted 1 mm anterior to the bregma, 2.5 mm right of the midline, and 4 mm below the surface of the skull. The guide cannula was sealed with a stainless steel wire of the same length. The rats were allowed at least 5 days to recover from surgery. On the day of the experiments, the inner steel wire was replaced by a 30-gauge stainless steel injection cannula (0.15 mm inner diameter, 12.5 mm in length) that protruded 2.5 mm beyond the guide cannula. Throughout the experiments, rats were randomly divided into four groups (n=4~5 per group): vehicle + saline, vehicle + cocaine, drug + saline, and drug + cocaine. Drugs were infused unilaterally into the central part of the right dorsal striatum 5 min prior to cocaine or saline injection in a volume of 1 µl at a rate of 0.2 µl/min in freely moving rats. The progress of the injection was monitored by observing the movement of a small air bubble along the length of a precalibrated PE-10 tubing inserted between the injection cannula and a 2.5 µl Hamilton microsyringe. After the completion of the injection, the injector was left in place for an additional 5 min to reduce any possible backflow of the solution along the injection tract. Cocaine (Belgopia, Louvain-La-Neuve, Belgium) was dissolved in physiological saline (0.9% NaCl), and each rat received a single systemic injection of cocaine (20 mg/kg, i.p.). Parallel to each drug injection, dimethylsulfoxide (DMSO)/artificial cerebrospinal fluid (aCSF) containing (mM) 123 NaCl, 0.86 CaCl₂, 3.0 KCl, 0.89 MgCl₂, 0.50 NaH₂PO₄, and 0.25 NaH₂PO₄ aerated with 95% O₂/5% CO₂, pH 7.2~7.4 or NaCl was injected into either the center of the dorsal striatum or the peritoneum 5 min before cocaine or saline injection in each experiment, as a control. All drugs except cocaine were purchased from Tocris Cookson (Ellisville, MO, USA), and the drug solutions were adjusted to pH 7.2~7.4 with 1 N NaOH, if necessary. Drug concentrations were determined based on previous studies (Ahn et al., 2007; Lee et al., 2008; Kim et al., 2009).

The NO microbiosensor was prepared as previously described (Alvin et al., 2008) and was cleaned by cycling the applied potential between +1.4 and -0.2 V for 10 cycles at a scan rate of 500 mV/s in 0.5 M H₂SO₄ solution, followed by washing with distilled water. Subsequently, the Pt microelectrode was coated with a conductive polymer (CP) through an electropolymerization reaction with a carboxylic acid group TTCA monomer in a 0.1 M TBAP/CH₂Cl₂ solution. This was accomplished by cycling the potential between 0.0 and 1.5 V two times at a scan rate of 50 mV/s. The electrode was then washed with CH₂Cl₂ to remove the excess monomer. The CP-coated Pt microelectrode was immersed for 12 h in 0.1 M phosphate buffered saline (PBS; pH 7.0) containing 20 mM 1-ethyl-3 [3-(dimethylamino)-propyl] carbodimide (EDC) to activate the CP carboxylic acid groups. At this point, the EDC treated CP-modified microelectrode was washed with buffer solution and subsequently incubated for 48 h in 5 mM PBS solution containing 6 mg/mL cytochrome c (cyt c) at 4℃. Using this procedure, cyt c was covalently bound through its amine groups to the carboxylic groups on the poly-5,2':5',2-terthiophene-3'-carboxylic acid (poly-TTCA), thus forming amide bonds. The cyt c/poly-TTCA microelectrode was dipped in 1% Nafion solution (diluted with ethanol) for 2 min. The Nafion film was then dried for 1 h in a calcium chloride atmosphere. Nafion films were dried in a low-humidity atmosphere provided by calcium chloride pellicles in a sealed container, which increased stability.

The real-time detection of NO was performed previously described (Alvin et al., 2008; Lee et al., 2010). Briefly, rats were anesthetized with 8% chloral hydrate and placed in a Stoelting stereotaxic apparatus. The enzyme-coated electrode and the reference electrode of the NO microbiosensor were inserted under ascetic conditions at the coordinates of 1 mm anterior of the bregma, 2.5 mm right of the midline, and 4 mm below the surface of the skull where the drugs were infused. The sensor electrode was connected to a Potentiostat/Galvanostat (Model PT-1; Kosentech, Pusan, Korea), which measured and recorded the electrochemical signal. The sensor was calibrated with a series of NO standard solutions during pre- and post experimental measurements. A 20-min time point was selected based on a time course of cocaine injection with peak NO levels at 20 min.

Data from NO measurements were recorded as currents which were converted to concentrations (µM). Statistical significance between groups was determined using one-way ANOVA followed by a Tukey's honestly significant difference test in GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA). The level of statistical significance was set to p < 0.05.

The NO microbiosensor was calibrated before and after measurement of NO levels in the series of experiments (Fig. 1A). Under optimized conditions, the steady-state currents exhibited a linear relationship with NO concentrations in the range of 0~55.0 µM (Fig. 1B). The sensitivity of the NO microbiosensor was 0.117 ± 0.006 µA/µM. The detection limit of NO concentration was 13 ± 3 nM based on five replicates to determine the standard deviation of the blank noise (95% confidence level, k = 3, n = 5). The Nafion film had a pore size less than 50 nm, which prevents the diffusion of peroxide and oxygen. However, NO gas can diffuse somewhat easily through Nafion. In addition, the Nafion layer prevents microelectrode fouling due to the nonspecific adsorption of proteins and other biological materials present in the brain. To remove the interference from oxygen, superoxide, and hydrogen peroxide, a thin Nafion film was coated onto the cyt c/poly-TTCA surface of the electrode. The selectivity of this modified electrode was evaluated with cyclic voltamograms in the presence of oxygen and other reactive oxygen species, such as hydrogen peroxide and superoxide. The sensor exhibited no significant response to these electroactive species.

To determine whether acute injection of cocaine alters the NO efflux in the dorsal striatum, we measured real-time amperometric NO responses for 120 s before and 5 min after acute saline or cocaine injection up to 60 min at 10 min time intervals. Acute systemic injection of cocaine increased NO levels compared with saline injection (Fig. 2A and 2B). Semiquantitation confirmed that as a result of cocaine, but not saline, injection, NO levels were significantly increased at 10 min, remained up to 20 min, and then returned to basal levels (Fig. 2C).

Because acute cocaine increased NO levels, this experiment was conducted to determine the involvement of dopamine receptors in the regulation of NO efflux in the dorsal striatum. Intrastriatal infusion of the dopamine D1 receptor antagonist, SCH23390 (7.5 nmol), significantly decreased an acute cocaine-evoked increase in NO levels (Fig. 3A). Intrastriatal infusion of the dopamine D2 receptor agonist, quinpirole (5 nmol), also significantly decreased NO levels (Fig. 3B). However, SCH23390 or quinpirole alone did not alter NO levels compared with the saline-injected group (Fig. 3).

Because dopamine D1 receptor stimulation interacts with NMDA receptors in the ventral tegmatal area (Schilström et al., 2006), this experiment was conducted to determine the involvement of NMDA receptors in the regulation of NO efflux after acute cocaine administration. Intrastriatal infusion of the NMDA receptor antagonists, MK801 (2 nmol) and AP5 (2 nmol), significantly reduced the acute cocaine-evoked increase in NO levels (Fig. 4). However, MK801 or AP5 alone did not alter NO levels compared with the saline-injected group (Fig. 4). Changes in real-time NO levels caused by the drug injection in all experiments are summarized in Table 1.