In vivo Microdialysis Experiment

Figure 1A illustrates the sequence of experimental events. Following recovery from surgery, microdialysis rats were placed in the test chambers for two, 3.5-hour (h) habituation sessions (one/day). Their catheters were tethered to the i.v. drug infusion line and their cannulae were tethered to the steel spring casing used to protect the microdialysis tubing during sampling. During each habituation session, each rat received 78 μL of saline i.v. over 5, 45 and 90 s, with one infusion given every 90 minutes in counter-balanced order. We injected a volume of 78 μL because for cocaine injections, we would inject 34 μL of saline to account for average catheter volume + 10 μL cocaine solution + 34 μL of saline to ensure that none of the cocaine remains in the catheter. Catheter patency was verified on the second habituation day by i.v. infusion of the short-acting barbiturate, sodium thiopental (20 mg/mL in sterile water, 0.1–0.2 mL/rat; CDMV, St-Hyacinthe, QC). All rats became ataxic within 5 s of the infusion, confirming catheter patency. In rats, sodium thiopental has a T_1/2 of ~4–5 h (Shideman et al., 1953), thus we do not expect it to influence microdialysis measurements performed on the next day. On the following day, rats were placed in the test chambers and dialysis probes were lowered into the striatum for a 5-h habituation period. During this period, aCSF was perfused through the microdialysis probe at a rate of 2.0 μL/minute. Rats were awake and freely moving during the experiment. Three hours and fifty minutes into the habituation period, animals were tethered to the cocaine infusion line. It contained 10 μL of cocaine solution (Medisca Pharmaceutique Inc, St-Laurent, QC; 2 mg/kg/infusion in 0.9% physiological saline) separated from additional saline by a small air bubble. The other end of the line was attached to a 3 c.c. BD syringe placed on a syringe pump. A 2 mg/kg dose of cocaine is similar to doses used in prior studies that have measured cocaine or dopamine concentrations in the striatum using in vivo microdialysis (Hurd et al., 1988; Hurd & Ungerstedt, 1989; Ferrario et al., 2008). This dose also evokes robust immediate early gene expression in the dorsal striatum (Samaha et al., 2004).

Panel a illustrates the timeline of experimental events for the in vivo microdialysis study. Rats were implanted with a unilateral cannula into the dorsal striatum and a catheter into the jugular vein. Following recovery, the rats were habituated to the in vivo microdialysis apparatus and to the i.v. infusion procedure on 2 daily sessions. On the following test day, microdialysis probes were inserted into the dorsal striatum and i.v. catheters were tethered to the cocaine infusion set up. Each rat received an i.v. infusion of 2 mg/kg cocaine, delivered over 5, 45 or 90 s, in counterbalanced order, with infusions administered 90 minutes apart. Dialysate samples were collected every minute for 5–10 minutes before each infusion and for 15 minutes thereafter. Panel b shows the sequence of experimental events for the psychomotor activity study. Rats were implanted with an intrajugular catheter and allowed to recover for 7 days. Rats were then habituated to the psychomotor activity cages and i.v. infusion lines on 2 daily sessions. On the following test day, rats were tethered to the cocaine infusion lines and locomotor activity was measured. Each rat received i.v. cocaine (2.0 mg/kg/infusion) delivered over 5, 45 and 90 s, in counterbalanced order, with infusions administered 90 minutes apart.

Over the last 10 minutes of the 5-h habituation period, 10 baseline dialysate samples were collected, at 1-minute intervals and at a flow rate of 2.0 μL/minute, yielding 2.0 μL/sample. Next, each animal received the first of three i.v. cocaine infusions (2.0 mg/kg/10 μL/infusion), delivered over 5, 45 and 90 s, injected 90 minutes apart, in counter-balanced order. Cocaine infusions were spaced 90 minutes apart because this is 3–4 times longer than cocaine’s T_1/2 in rat brain (Nayak et al., 1976; Hurd et al., 1988). Thus, the 90-minute inter-infusion interval reduces the possibility of carry-over effects between infusions [see also (Hurd et al., 1988)]. Following each cocaine infusion, 15 samples were collected, at 1-minute intervals. Five 1-minute baseline samples were also collected prior to the 2^nd and 3^rd cocaine infusions. Samples were collected in a 300-μL polypropylene microsampling vial placed on the end of the outlet line. Samples were immediately placed in dry ice and stored at–80°C until processing. At the end of sampling, each microdialysis probe was visually inspected for leaks or breakage. None were detected.