Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 300–350 g were used in this study. Animals were maintained on a 12:12 light/dark schedule. All experiments were performed during the light cycle. Water and food was provided ad libitum. All animal use procedures were performed in an AAALAC accredited facility in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Yale University Animal Care and Use Committee.
All reagents for the HPLC mobile phase and the perfusion fluid were of an analytical grade and were obtained from Sigma (St. Louis, MO). PCP hydrochloride was purchased from Sigma. Clonidine hydrochloride and guanfacine hydrochloride were obtained from RBI (Natick, MA), and BRL-44408 maleate was purchased from Tocris-Cookson (Ellisville, MO). All drugs were dissolved in 1 ml/kg of isotonic sterile saline.
4.3. Surgical preparation
Animals were given 1 week to acclimate after arrival in the vivarium before undergoing probe implantation. All rats were anesthetized with halothane (1–5%, as needed) and placed in a Stoelting stereotaxic instrument fitted with blunt ear bars. A thermostatically controlled electric heating pad was used to maintain the body temperature at 37 °C.
An incision (~10 mm) was made in the skin over the skull and the wound margin was infiltrated with 2% lidocaine. Burr holes were drilled for two skull screws and two concentric microdialysis probes. The probes were constructed using Hospal AN69 polyacrylonitrile dialysis tubing (Renal Care, Lakewood, CO). The outer diameter of the probes was 330 μm, and the exposed membrane tip was 3.0 mm.
The probes were positioned bilaterally in the right and left medial prefrontal cortex, respectively: AP, +3.2; L, 1.2 angled 10° towards midline; V, −5.6 and AP, +3.2; L=0.7 no angle; V, −5.0 with respect to bregma (in mm) according to the atlas of Paxinos and Watson (1982). Loctite Tak Pak 444 (Rocky Hill, CT) was used to initially secure probes and screws to the skull before removing the stereotaxic probe holders. Dental cement was then used to build up a protective cap around the microdialysis probes.
Immediately after surgery the animals were connected to a Harvard syringe pump (Harvard Apparatus, Holliston, MA) via fused-silica tubing (Polymicro Inc., Phoenix, AZ) encased in PE50 polyethylene intramedic tubing and a swivel connector that was attached to a liquid swivel/balance arm assembly (Instech Laboratory, Plymouth Meeting, PA). During the recovery period, probes were perfused at a rate of 0.5 μL/min with a Ringer’s solution containing 145 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, and 1.2 mM CaCl2. The animals were allowed to recover in a modified home cage 24 h prior to collection of the dialysis samples with food and water available.
4.4. Microdialysis procedure
On the day of the experiment, the flow rate was increased to 2 μL/min approximately 2 h before beginning the collection of baseline samples. Dialysates were collected every 20 min; after 4 baseline samples were collected, animals were pretreated with an intra-peritoneal (i.p.) injection of either 0.9% saline (the vehicle), clonidine (0.0033, 0.01 or 0.05 mg/kg) or guanfacine (0.05 or 0.5 mg/kg), before receiving an injection of PCP (2.5 mg/kg, i.p.) 20 min later. In a separate study, BRL (1.0 mg/kg) was administered 20 min prior to clonidine. In addition, for some control experiments, the animals only received one injection of saline, clonidine (0.01 or 0.05 mg/kg), guanfacine (0.5 mg/kg) or BRL (1.0 mg/kg). The doses of clonidine, guanfacine and PCP were based on our previous studies (Jentsch et al., 1998a
; Jentsch and Anzivino, 2004
; Marrs et al., 2005
). The dose of BRL was chosen following a preliminary dose-response study, in which 1 mg/kg was found to have no significant effect on extracellular PFC dopamine levels, yet to be effective at blocking the effect of clonidine on PCP-induced elevation of dopamine levels. In addition, a 1 mg/kg dose of BRL has been found by others to exert selective alpha2A antagonism (Galeotti et al., 2004
After each 20 min sample was collected, the dialysate was injected immediately onto a high pressure liquid chromatography (HPLC) system equipped with electrochemical detection for analysis of dopamine content. Each HPLC system used a narrow-bore column (2.0 mm i.d.; 3 μm C-18 particles, Keystone, Bellefonte, PA) and Bioanalytical Systems LC-4C potentiostats (West Lafayette, IN). The Eapp was +0.55 V versus an Ag/AgCl reference electrode. The mobile phase consisted of 0.l M NaH2PO4, 450 mg/L of octylsulfonic acid, 7.2% (vol/vol) acetonitrile, 0.25% EDTA and 350 μl/L of triethylamine (pH 5.0). A detection limit of 5 fmol for dopamine was routinely achieved with this system.
After the termination of each experiment, animals were anesthetized with chloral hydrate (600 mg/kg, i.p.) and perfused transcardially with 0.9% saline. The brains were removed and post-fixed in 10% formalin before being placed in sucrose (30% in saline) for 2 days. Serial sections were cut on a freezing stage microtome at 250 μm intervals and stained with Cresyl Violet. Probe placement was verified for all the animals used in this study.
4.6. Data analysis
For the microdialysis data, a within-group analysis was performed by using one-way ANOVA, with time as the repeated measure. Comparisons between drug-treated and control groups were analyzed using a two-way ANOVA with time as the repeated measure. When a significant F value was obtained, further comparison of individual data at corresponding time points between groups were carried out using Tukey’s post hoc tests.