Materials and methods ing tubing connections. This was performed rapidly and

52 E specific role of cortical 5-HT receptors in the modulation before the start of the experiments, microdialysis probes 2A of mesocortical DA function. were lowered carefully through the guide cannulae into In the present study, we examined the role of pre- awake rats and secured in place with Krazy Glue Gel E. frontocortical 5-HT receptors through the use of the Immediately following probe implantation, animals were 2A highly selective antagonist M100,907 R-1-a-2,3- placed in clear Plexiglas test chambers and tethered to dimethoxyphenyl - 1 - [2 - 4 - fluorophenylethyl] - 4 - piper - counterbalance arms that permitted relatively free move- idine-methanol. This ligand has 100 fold selectivity for ment. They remained there with food and water until the the 5-HT Ki50.36 nM over the 5-HT Ki5105 nM start of the experiments. 2A 2C receptor [25] and has negligible affinity for DA receptors A micro-infusion pump PHD 2000 E, Harvard Ap- Ki.540 nM [14]. M100,907 was infused directly into paratus and liquid swivels were used to perfuse the buffer the mPFC through reverse microdialysis. Effects on both through the probes at a constant rate. Although each 1 basal and potassium K -stimulated DA release were experiment is self-contained with its own appropriate examined. Additionally, the effects of intracortical controls, perfusion flow rates differed between some M100,907 on alterations in DA release produced by the experiments. An initial experiment Experiment 2 em- systemic administration of 1-2,5-dimethoxy-4- ployed a flow rate of 1.5 ml min. In order to increase the iodophenyl-2-aminopropane DOI, a 5-HT agonist, recovery of DA, the flow rate was decreased to 1.0 ml min 2 were investigated. We hypothesized that intracortical infu- in all subsequent experiments. Dialysate samples were sion of a selective 5-HT antagonist would increase DA collected every 30 min until basal DA concentrations were 2A release in the mPFC. stable for at least 3 baseline samples. Drugs or artificial cerebrospinal fluid aCSF buffers with altered ionic compositions were then administered by manually switch-

2. Materials and methods ing tubing connections. This was performed rapidly and

flow rate and collection volumes were maintained. Sample 2.1. Animals and surgery collections then continued every 30 min for another 2.5 to 3.5 h depending on the experiment. Male Sprague–Dawley rats Zivic Miller, Hillson, PA, For experiments examining the effects of M100,907 on USA, weighing from 200 to 400 g at the time of surgery, basal or DOI-induced DA release Experiments 2 and 4, a were used throughout this study. Rats were housed in pairs modified, commercially available, aCSF was employed: in a temperature-controlled room on a 12 12 light dark Dulbecco’s phosphate buffered saline 137 mM NaCl, 2.7 cycle. Food and water were available ad libitum. Prior to mM KCl, 0.5 mM MgCl , 1.5 mM KH PO , 8.1 mM 2 2 4 surgery, the rats were anesthetized with a mixture of Na HPO , pH: 7.4. CaCl 1.2 mM and glucose 10 2 4 2 ketamine 70 mg kg and xylazine 6 mg kg injected mM were added to this solution. All previous work in the i.m., and then mounted in a stereotaxic frame. After dura present laboratory has employed this buffer which has was removed, 21 gauge stainless steel guide cannulae were excellent pH stability and produces stable DA levels in 1 chronically implanted on the brain surface above the control animals. However, the high K studies Experi- mPFC AP 3.2, ML 0.8 [27]. The guide cannulae were ments 1 and 3 required an increased KCl concentration secured to the skull with three skull screws covered with 80 mM and, to maintain solution osmolarity, a simulta- cranio-plastic cement. Animals were then housed indi- neous decrease in NaCl to 60 mM. Both adjustments vidually for the 3–5 day period between surgery and were not possible using the Dulbecco’s buffer solution. microdialysis experiments. Thus, a laboratory prepared Krebs–Ringer buffer 137 mM Each rat was used once, and after the experiment was NaCl, 3 mM KCl, 1.2 mM MgSO , 0.4 mM KH PO , 1.2 4 2 4 concluded, probe placements were verified histologically. mM CaCl and 10 mM glucose, pH: 7.4 was employed. 2 Only animals whose probe placements were verified to be The ionic concentrations of this normal Ringers was then 1 in the mPFC see below were used in the study. All modified KCl 80 mM, NaCl 60 mM to create a high K animal use procedures were in strict accordance with the Ringers solution. 1 NIH Guide for the Care and Use of Laboratory Animals For high K experiments, drug treated rats were pre- and were approved by the local animal care committee. treated for 30 min with the appropriate concentration of M100,907 dissolved in normal Ringer’s. This was fol- 2.2. Microdialysis lowed by 30 min perfusion with the drug dissolved in the 1 high K Ringers solution. During these experiments, Microdialysis probes of a concentric flow design were control rats received infusions of normal Ringers for 30 1 used [43]. Probes were constructed to dialyze the mPFC min followed by high K Ringers for 30 min. from the dorsally located anterior cingulate cortex, through the prelimbic cortex, and including the ventrally located 2.3. Drugs infralimbic subregion. The active dialyzing surface of the membrane Spectra Por Hollow, MW cutoff513 000, M100,907 Hoechst Marion Roussel, free base was diameter5200 mm was 5.0 mm in length. At 18 to 24 h administered intracortically via reverse dialysis. Solutions E .A. Pehek et al. Brain Research 888 2001 51 –59 53 were made by dissolving the drug in 2.5 ml of glacial h of perfusion and sample collection without drug. The acetic and 1 ml deionized water. aCSF was then utilized to vehicle group was perfused with aCSF throughout. dilute this solution to the appropriate concentrations 100 nM, 1.0 mM, 10 mM, and 100 mM, pH 7.4. 6-DOI- 2.5.3. Experiment 3 hydrochloride Research Biochemicals Incorporated, MA, USA was administered subcutaneously s.c. DOI 2.5 2.5.3.1. Effects of lower concentrations of M100,907 on mg kg or vehicle deionized water were injected in 1.0 DA efflux. Experiment 3A examined the effects of 1.0 mM 1 ml kg volumes. M100,907 on K -stimulated DA efflux. Drug treated rats 1 were compared to high K controls. Procedures were the same as in Experiment 1. Experiment 3B determined the 2.4. Chromatography effects of lower concentrations of M100,907 on basal dialysate DA. Following the collection of stable baselines, DA content of dialysate samples was measured by 100 nM concentrations were infused for 2 h. This was HPLC coupled with electrochemical detection. Twenty- followed by perfusion with 1.0 mM concentrations for 2 h microliter dialysis samples were injected onto a 2 mm in the same rats. Phenomenex column Ultracarb E, 3 mm particle size, ODS 20. The mobile phase consisted of 32 mM citric 2.5.4. Experiment 4 acid, 54 mM sodium acetate, 0.074 mM EDTA, 0.215 mM octylsulfonic acid, and 3 methanol vol vol, pH 4.2. To 2.5.4.1. Effects of DOI and M100,907 on cortical DA maintain separation of DA from its metabolites and 5- release. This study examined the effects of a systemic hydroxyindoleacetic acid, the pH of the mobile phase and injection of DOI on DA efflux in the mPFC. The receptor the concentration of octylsulfonic acid were adjusted as specificity was examined by investigating the ability of needed. A BAS LC-4C electrochemical detector with a intracortical infusions of M100,907 10 mM to attenuate glassy carbon electrode, maintained at a potential of 10.60 the effects of DOI. Four groups of rats were utilized in this V relative to an Ag AgCl reference electrode, was em- study: DOI alone, vehicle, M100,907 alone, M100,9071 ployed. The limit of detection for dopamine was 0.1 pg 20 DOI. The first group received a s.c. injection of DOI while m l. the second group received vehicle injections. A third group was perfused intracortically with 10.0 mM M100,907 for 3 h. The last group received similar M100,907 infusions plus 2.5. Experimental design a systemic injection of DOI. DOI was administered 30 min after the start of perfusion with M100,907. 2.5.1. Experiment 1 2.6. Data analysis Data were expressed, analyzed, and graphed as the 1 2.5.1.1. Effects of M100,907 on K -stimulated cortical percentage of the last 3 baseline samples. Statistical DA release. This experiment tested the ability of a high analyses were performed using repeated measures 1 K infusion to increase extracellular DA in the mPFC. ANOVAs. For two-way ANOVAs, time was the repeated 21 Furthermore, it tested the Ca -dependency of this effect measures factor and drug condition was the independent 1 and the ability of M100,907 to modulate the K -evoked factor. For one-way ANOVAs, time was the repeated release. Baseline samples were collected from all rats factor. Post-hoc comparisons employed Dunnett’s test for utilizing normal Ringer’s. One group of rats then received comparing treatment means with a control value. 1 21 infusions of a high K buffer with normal Ca 1.2 mM 21 while a separate group was perfused with a Ca -free high 1 K solution. Two other groups were pretreated with either

3. Results