Twenty-seven male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) weighing 225–250 g at the start of the study were used in accordance with guidelines mandated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were singly housed on a 12 hr reverse light-dark cycle and given food and water ad libitum. Unless stated otherwise, chemicals were purchased from Sigma (St. Louis, MO).
Implantation of self-administration catheters and tracer injections
The rats were deeply anaesthetized by intraperitoneal injections of a cocktail consisting of 45% ketamine (100mg/ml), 35% xylazine (20mg/ml) and 20% saline at a dose of 0.16 ml/100g of body weight. A silastic catheter (20 μl dead volume) was inserted via the external jugular vein into the right atrium of the heart, passed under the skin and fixed in the midscapular region. Immediately after the catheters were implanted, the rats were placed into a Kopf stereotaxic instrument and the retrograde tracer Fluoro-Gold (FG; Fluorochrome, Inc., Englewood, CO; 1% in 0.1M cacodylate buffer, pH 7.4) was injected iontophoretically into the VTA through 1.0 mm filament-containing glass pipettes pulled to tip diameters of 15–20 μm using 1 μA positive pulses (7 s on and 7 s off for 15 minutes). After surgery, the rats were kept warm until they awakened. Subsequently, the catheters were flushed daily with sterile saline to prevent clogging.
Self-administration of cocaine and vehicle; investigator-administered cocaine
Immediately following surgery and daily thereafter the rats were gently handled in order to flush the catheters with saline. One week after surgery, the animals were placed in a self-administration chamber (Med. Associates, St. Albans, VT) and allowed to self-administer cocaine (SAC group; n=9; 500 μg/kg per 30 μl infusion) or saline (SAS group; n=9) for six consecutive days (2h/day, during the dark part of the dark/light cycle) as previously described (Marinelli et al., 2003
). Briefly, nose-poking in the active hole delivered an infusion (cocaine or saline) and, for a period of 30 sec, illuminated the hole with an LED light, which in and of itself is reported to stustain a moderate level of responding (Marinelli et al., 2003
). Nose poking in the inactive hole had no consequences. Self-administration was regarded as established when nose pokes were significantly more numerous in the active than inactive hole. Rats given investigator-administered infusions of cocaine (YAC group; n=9) were placed in separate chambers located in the same environment as the SAC and SAS rats. These rats received infusions administered by the investigator in numbers and at time intervals reflecting the averages delivered on the same day to the rats in the SAC group. Catheters were to be regarded as having remained patent throughout the study if 200 μl of the ketamine/xylazine mixture could be delivered through them on the last day of the experiment. Thus, rats that did not succumb within 3–5 s following this treatment were to be removed from the study. All of the lines remained open in the present study. Self-administration cages were also equipped with two photocell beams located in the long axis of the cage to monitor locomotor activity.
Fixation of brains and immunocytochemistry
One and one-half hours after the beginning of the sixth daily self-administration session, the rats were deeply anesthetized and perfused transaortically with buffered (0.1 M Sorensen's phosphate buffer, pH 7.4 - SPB) aldehydes and the brains were sectioned and processed for immunohistochemistry as has been described [see Geisler and Zahm (2005)
for details]. Five adjacent series of sections were collected, each representing an entire brain from frontal pole to caudal medulla at intervals of 250 μm. Each series of sections was stored in a separate glass vial at −20°C in a cryoprotectant consisting of SPB containing 30% sucrose and 30% ethylene glycol.
One series of sections from each case was immersed in SPB containing 0.1% Triton X-100 (SPB-t) and a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 4–17 of human c-Fos at a dilution of 1:5000 (Anti-c-Fos [Ab-5] [4–17] Rabbit pAb, formerly from Oncogene Science, Cambridge MA, now from Calbiochem, San Diego CA). The following day, after thorough rinsing in SPB-t, the sections were immersed in SPB-t containing a biotinylated antibody made in donkey against rabbit IgGs at a dilution of 1:200 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for an hour. Afterward, the sections were rinsed in SPB and then immersed in SPB containing avidin-biotin-peroxidase complex (ABC) at a dilution of 1:200 (Vector Laboratories, Burlingame CA) for an hour. After thorough rinsing in SPB, the sections were reacted in 0.025 M Tris buffer (pH 8.0) containing 0.015% 3,3’–diaminobenzidine (DAB), 0.4 % nickel ammonium sulfate and 0.003 % hydrogen peroxide, which generates an insoluble black reaction product.
The sections were then subjected to additional rinsing in SPB and then immersed in SPB-t containing an antibody raised in rabbit against FG and used at a dilution of 1:5000 (Chemicon, Temecula, CA, USA). The following morning the sections were rinsed in SPB-t and immersed for one hour in SPB-t containing a donkey antibody against rabbit IgGs used at a dilution of 1:200 (Jackson). Following further rinsing in SPB the sections were immersed for one hour in SPB containing rabbit peroxidase-anti-peroxidase (PAP) at a dilution of 1:3000 (ICN Biomedicals, Inc., Aurora, OH), after which they were again rinsed thoroughly. Then the sections were immersed for 20–30 min in 0.05 M SPB (pH 7.4) containing 0.05% DAB, 0.04% ammonium chloride, 0.2% β-D-glucose, and 0.0004% glucose oxidase, which generates an insoluble brown reaction product. The sections then were mounted onto gelatin coated slides, dehydrated through a series of ascending concentrations of ethanol, transferred into xylene, and coverslipped with Permount (Fisher, Pittsburgh, PA). Staining for Fos and FG was absent when the relevant primary or secondary antibodies, ABC or PAP reagents were omitted.
Additional sections were prepared to exhibit single labeling for tyrosine hydroxylase (TH)-ir (Chemicon), for the evaluation of FG injection sites, and for double-labeling with Fos-ir and TH-ir or Fos-ir and nitric oxide synthase (NOS)-ir (Sigma), in order to determine if Fos-ir was expressed in dopaminergic and cholinergic neurons, respectively. Protocols to accomplish this were carried out as described above using appropriate second antibodies, except, for Fos/TH double-labeling, the second antibodies were conjugated to fluorescent compounds fluorescein and Texas Red and examined with epifluorescence illumination (Jackson ImmunoResearch Labs). All of the antibodies used in the study have been well characterized by the manufacturers and were evaluated in our hands with appropriate omission controls.
Analysis of Fos-ir in VTA afferents
The analysis was carried out with the aid of the MDPlot5 hardware-software platform (Accustage, Shoreview, MN), which was used to generate digitized 2-D representations of the outlines of the sections, ventricles, and major white matter bundles, as well as all FG-ir (retrogradely labeled) and double-labeled (FG-ir plus Fos-ir) neurons. For forebrain, series of 17 consecutive sections spaced at 250 μm intervals beginning at the rostral tip of the accumbens and extending to include the rostral one-third of the lateral hypothalamus were analyzed. The lateral habenula was evaluated in a separate series of sections. For brainstem, similarly spaced series of six sections beginning at the midpoint of the interpeduncular nucleus and extending caudalward to the level of the laterodorsal tegmental nucleus were evaluated. Structures of interest containing retrogradely labeled neurons thus were equivalently represented in all of the evaluated cases. FG-ir and double-labeled neurons were counted with the aid of an MDPlot5 software feature and double-labeled neurons were expressed as percent of retrogradely labeled neurons. Means reflecting 4 cases for each of the three experimental groups (SAS, SAC and YAC) were tested with a one-way ANOVA followed as indicated by Fishers LSD post hoc test.