Animals

Adult, male, Sprague–Dawley rats (230–250 g) were group-housed, but after icv and intra-nuclei (CeA, BNSTl or AcbSh) cannulation, they were housed individually. All animals had free access to food (Lipton, India) and drinking water except during the test period or some special experimental protocols. They were maintained on a 12 h dark/light cycle, in controlled temperature (25±2°C) and relative humidity (50–70%). The experimental procedures were approved by the Institutional Animal Ethical Committee.

Intracerebral Cannulations

The detailed procedure of cannulation, drug administration, and post-surgical care has been described (Kokare et al, 2005, 2006). A stainless steel guide cannula (C316G/Spc; Plastics One, Roanoke, VA) was implanted into the right lateral cerebral ventricle using the stereotaxic coordinates, −0.8 mm posterior, +1.3 mm lateral to midline, and 3.5 mm ventral with respect to bregma (Paxinos and Watson, 1998). Similarly, the cannulae were implanted bilaterally in the CeA (−2.4 mm posterior, ±4.0 mm lateral to midline and 8.0 mm ventral), BNSTl (−0.3 mm posterior, ±1.6 mm lateral to midline and 6.0 mm ventral) or AcbSh (+1.7 mm anterior, ±0.8 mm lateral to midline and 6.5 mm ventral) in other groups. The animals were allowed a recovery period of 7 days and those with any neurological/motor deficits were excluded from the study.

Ethanol Treatment

The rats were given ethanol in a liquid diet for 15 days according to the previously described protocol (Pandey et al, 1999; Kokare et al, 2006). Briefly, rats were given a nutritionally balanced control liquid diet (Novartis, India) for 2 days for adaptation to the novel food. From day 3 onwards, ethanol was gradually introduced into the liquid diet starting with 4.5% (v/v) ethanol on the first day, 7.5% (v/v) ethanol on the second day and 9% (v/v) ethanol thereafter for 15 days (ethanol-fed group, n=27). The rest of the animals continued on the nutritionally balanced control liquid diet (pair-fed control group, n=23). A fresh aliquot of ethanol diet or control liquid diet (100 ml/rat) was provided each morning. After 15 days, all the animals were placed on an ethanol-free, nutritionally balanced diet and killed at 0, 24, 48, and 72 h post-withdrawal, and their brains processed for CART immunolabeling.

Icv, Intra-CeA, -BNSTl and -AcbSh Administration of CART and CART Antibody

The methods of icv and nuclei-specific injections and preparation of artificial CSF (aCSF) have already been described (Kokare et al, 2005, 2006). Cannulated rats were randomly allocated to different groups (n=8 in each). All injections were performed between 0900 and 1200 h in a randomized way and rats, once used, were not re-employed. CART (54–102) was dissolved in aCSF and injected into the right lateral ventricle or bilaterally into the CeA, BNSTl or AcbSh. Various agents like aCSF (5 μl/rat, icv; n=7, and 1 μl each side for intra-CeA, -BNSTl or -AcbSh; n=6, n=7 or n=7 respectively) and CART (25–100 ng/rat, icv; n=8 per group and 2–20 ng/rat for intra-CeA, -BNSTl or -AcbSh; n=6, n=8 or n=6 per group respectively) were given as single injection and subjected to the social interaction test (see below) following an interval of 30 min.

To study the effect of immunoneutralization of endogenous CART on social interaction following ethanol-withdrawal, cannulae were implanted either into the lateral ventricle or bilaterally into the CeA, BNSTl or AcbSh as described above. Twenty-two hours following withdrawal, rats were injected with (i) aCSF (5 μl/rat, icv; n=8 or 1 μl each side for intra-CeA, -BNSTl or -AcbSh; n=6 in each group), (ii) non-immune serum (1 : 500 dilution, 5 μl/rat, icv; n=7 or 1 μl each side for intra-CeA, -BNSTl or -AcbSh; n=7 in each group) or (iii) antibody against CART (1 : 500 dilution, 5 μl/rat, icv; n=6 or 1 μl each side for intra-CeA; n=8, -BNSTl; n=8 or -AcbSh; n=6). Two hours after the injection, animals were subjected to social interaction test (see below). Earlier studies used the antibodies for immunoneutralization in similar timeframe and dilution range (Scruggs et al, 2003). The responses were scored by a trained observer blind to various treatment conditions. In all experiments, CART and CART antibody-treated groups were run in parallel with their respective control group. The placement of the cannula for icv injection was tested for accuracy by injecting dilute India ink and post-mortem examination of the distribution of ink in the ventricles (Kokare et al, 2006) and the 52 animals with correct placement were used for statistical analysis.

Following intra-CeA, -BNSTl and -AcbSh injections, the brains (168 rats) were removed, sectioned, stained with cresyl violet, and the position of the tip of each injection site was determined (see Supplementary Figure S1). Only animals with correct cannula placements (45 rats for intra-CeA, 52 rats for intra-BNSTl, and 44 rats for intra-Acb) were used for statistical analysis.

Social Interaction Test for Anxiety

The social interaction test (File and Hyde, 1978) was performed according to Gonzalez et al (1998) with slight modification. Two rats naïve to the test were taken. The index rat was implanted with cannula, while the untreated dummy partner was taken from separate cage and placed into the center of a test box (60 × 60 cm open field with 16 15 × 15 cm squares marked on the floor) simultaneously for a 10-min period. Reduced social interaction time reflected the increased anxiety-like behavior. Locomotor activity was measured as the number of squares entered during the interaction session. The time that the index rat socially interacted, which includes genital investigation, sniffing, following, grooming, kicking, crawling under or over the partner, and touching or nearly touching their faces with the untreated dummy partner was visually measured. The occurrence of at least one of the behaviors was recorded and the data are shown in Figures 1, 2, 3, 4 and 5.

Figure 1 Effect of different doses of CART (54–102) administered via icv or intra-CeA route on social interaction time (a and c) and number of crossovers (b and d) in the social interaction test in normal rats. The data were analyzed by one-way ANOVA with repeated measures on drug treatments followed by post hoc Dunnett's test. Each bar is the mean±SEM for 6–8 rats. *P<0.01 vs aCSF-treated group. Full size image

Figure 2 Effect of different doses of CART (54–102) administered bilaterally via intra-AcbSh or -BNSTl route on social interaction time (a and c) and number of crossovers (b and d) in the social interaction test in normal rats. The data were analyzed by one-way ANOVA with repeated measures on drug treatments followed by post hoc Dunnett's test. Each bar is the mean±SEM for 6–8 rats. No significant difference was found at the different doses of CART administered (P>0.05). Full size image

Figure 3 Effect of chronic ethanol treatment followed by withdrawal at 0, 24, 48, and 72 h time points on social interaction time. The data were analyzed by repeated-measure one-way ANOVA followed by post hoc Dunnett's test. Each bar is the mean±SEM for 5–8 rats. *P<0.05 or **P<0.001 vs pair-fed group. Full size image

Figure 4 Effect of aCSF, non-immune serum, and CART antibody administered by icv or intra-CeA route on the social interaction time (a and c) and number of crossovers (b and d) in pair-fed and/or 24 h ethanol-withdrawal rats. The agents were administered 2 h before the test. The data were analyzed by unpaired t-test or one-way repeated measures ANOVA followed by Student-Newman–Keuls test. Each bar is the mean±SEM for 6–8 rats. ***P<0.0002 and #P<0.0001 vs pair-fed group; *P<0.01 and **P<0.001 vs non-immune serum-treated group following 24 h ethanol withdrawal. Full size image

Figure 5 Effect of aCSF, non-immune serum and CART antibody administered by intra-AcbSh or -BNSTl route on the social interaction time (a and c) and number of crossovers (b and d) in pair-fed and/or 24 h ethanol-withdrawal rats. The agents were administered 2 h before the test. The data were analyzed by unpaired t-test or one-way repeated measures ANOVA followed by Student-Newman–Keuls test. Each bar is the mean±SEM for 6–8 rats. *P<0.0001 vs pair-fed rats. No significant difference was found in the non-immune serum-, and CART antibody-treated groups following 24 h ethanol withdrawal (P>0.05). Full size image

Immunocytochemistry

Brains of ethanol-treated as well as pair-fed rats collected at the 0, 24, 48, and 72 h post-withdrawal time points were processed for immunocytochemical labeling. Rats were anesthetized (pentobarbital, 50 mg/kg, intraperitoneally), perfused transcardially with heparinized phosphate-buffered saline (PBS; pH 7.4) for 30 s followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10–15 min. The brains were post-fixed in the same fixative overnight, cryoprotected in 30% sucrose solution, embedded, and serially sectioned on a cryostat (Leica) at 20 μm thickness in the coronal plane and collected in PBS. Sections were processed for CART immunolabeling using the streptavidin-biotin-peroxidase method described earlier (Singru et al, 2007). Briefly, sections were incubated in mouse monoclonal primary antibodies against CART (54–102) diluted in 1% bovine serum albumin (Sigma) containing 0.3% Triton X-100 and 0.09% sodium azide at 1 : 5000 dilution for 2 days at 4°C. After rinsing in PBS, the sections were incubated in biotinylated-IgG followed by ABC (Vector) and the immunoreaction product was developed in diaminobenzedine/H 2 O 2 in Tris buffer. Sections were dehydrated, cleared in xylene, and mounted with DPX. To ensure reliable comparisons among different groups and maintain stringency in tissue preparation and staining conditions, sections from the brains of different groups were processed at the same time under identical conditions. The details of generation of CART antibody and specificity have already been described (Thim et al, 1998). Omission of primary antibody and replacement with BSA produced no immunoreaction. In preadsorption controls, 1 ml diluted antibody incubated with CART at 10−5 M for 24 h before incubation completely blocked the immunoreaction.

Morphometric Analysis

The area occupied by CART-immunoreactive cells/fibers in the CeA, BNSTl and AcbSh was evaluated using microscopic images from predetermined areas in the sections. The images (× 480) were analyzed using Leitz-LaborLux S microscope, CCD video camera system (JVC, Japan) and Leica-Qwin Standard software (version 3). The method has already been standardized in our laboratory (Sakharkar et al, 2005).

The area (μm2) covered by CART immunoreactivity in the cells/fibers was estimated from the transverse sections passing through the CeA, BNSTl, and AcbSh from the pair-fed and ethanol-withdrawn rats. Figures 6 and 7 demarcate the rectangular area in each section from which the cell/fiber area was evaluated. The images of immunoreactive cells/fibers were digitized, the background was considered as threshold, and areas occupied by immunostained cells/fibers were measured based on individual pixel intensity in the pair-fed and rats subjected to ethanol withdrawal. The immunoreactive cells/fibers above the threshold were filled with overlaid color, and the area of the color overlay was automatically obtained using Leica-QWin Standard software. Five measurements from predetermined fields of the CeA, BNSTl and AcbSh on both sides of each brain were taken. The data from all the animals in each group were pooled separately and the mean±SEM calculated.

Figure 6 Photomicrographs showing CART-immunoreactive (CART-ir) fibers (arrows) in the CeA of pair-fed (a) and ethanol-withdrawn rats at 0 h (b), 24 h (c), 48 h (d), and 72 h (e). No significant difference in the CART immunoreactivity is seen in the CeA of pair-fed (a) and 0 h (b) ethanol-withdrawn rats. As compared to these animals, a dramatic increase in CART immunoreactivity is seen 24 h (c) following ethanol withdrawal. However, the CART immunoreactivity is considerably reduced in 48 h (d) and 72 h (e) ethanol-withdrawn animals; the immunoreactivity is comparable to that in the pair-fed animals. Diagram (f), represents the semiquantitative morphometric analysis of CART immunoreactivity in the CeA of pair-fed and ethanol-withdrawn animals at different time points. The outline of the transverse section through the brain (coordinates: bregma −2.56 mm, Paxinos and Watson, 1998) indicates the region of the CeA (square not to scale) from which the measurements were collated. BLA, lateral part of basolateral amygdaloid nucleus; MeAD, anterodorsal part of medial amygdaloid nucleus; opt, optic tract. *P<0.001 vs pair-fed group. Scale bar=50 μm. Full size image

Figure 7 Photomicrographs showing CART-immunoreactive (CART-ir) fibers (arrows) in the BNSTl and CART-ir cells (arrows) in the AcbSh of pair-fed (a, g) and ethanol-withdrawn rats at 0 h (b, h), 24 h (c, i), 48 h (d, j), and 72 h (e, k). CART immunoreactivity profile is similar in the brain sections of the pair-fed (a, g) and the rats at different time points post-ethanol withdrawal (b-e, h-k, P>0.05). Diagrams f and l represents the semiquantitative morphometric analysis of CART immunoreactivity in the BNSTl and AcbSh of pair-fed and ethanol-withdrawn animals at different time points. The outlines of the transverse sections through brain indicate the regions of the BNSTl and AcbSh at the coordinates −0.30 and +1.70 mm with reference to bregma respectively (Paxinos and Watson, 1998) from which the measurements were collated (square not to scale). ac, anterior commissure; AcbC, nucleus accumbens core; LSV, ventral part of the lateral septal nucleus; LV, lateral ventricle. Scale bar=50 μm. Full size image

Statistical Analyses

For the dose-dependent study of CART in the social interaction test, statistical significance was determined by one-way analysis of variance (ANOVA) with repeated measures on drug treatments followed by post hoc Dunnett's test. The results of ethanol-withdrawn rats at different time points were also analyzed by one-way ANOVA followed by Dunnett's test. The data obtained from ethanol-withdrawn and pair-fed rats were compared with the unpaired t-test. The data from immunoneutralization and morphometric studies were analyzed with one-way repeated measures ANOVA, and individual means were compared by the post hoc Student-Newman–Keuls test. Differences were considered significant at P<0.05.