Effect of gonadal/hormonal status on aromatase mRNA expression in different brain regions.

<p>Expression levels of the long form of aromatase were studied in the amygdala, BNST, POA, dorsal hippocampus, and cingulate cortex of castrated male, gonadally intact male, ovx female and ovx+E2 female rats. Each panel represents a different brain region. For each panel, qPCR amplification curves with relative fluorescent units (RFU) and the area punch dissected (red circle in the schematics of brain coronal sections, adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100628#pone.0100628-Paxinos1" target="_blank">[48]</a>) are shown at the top. C_T (cycle threshold) values on the amplification curve indicate the number of cycles required for the fluorescent signal to cross the threshold, which was set at 10 standard deviations above mean fluorescence generated during baseline cycles. Relative aromatase mRNA expression for each brain region is shown in bar graphs (ovx females set as 1.0) with representative images of agarose gels showing RT-PCR products for amplified aromatase (Aro) and GAPDH DNA fragments at the bottom. In the amygdala <b>(A)</b>, aromatase mRNA levels were affected by both sex (p = 0.006) and gonadal/hormonal status (p = 0.01), whereas in the BNST <b>(B)</b> and the POA <b>(C)</b>, gonadally intact males showed the highest aromatase mRNA expression, which was significantly different from all other groups; ** significant difference between intact males and other groups (p<0.01). In contrast, in the dorsal hippocampus <b>(D)</b> and cingulate cortex <b>(E)</b> aromatase mRNA levels did not differ among the groups.</p

mRNA sequence of rat aromatase.

<p>Exon boundaries in the rat mRNA sequence of cytochrome P450 (NCBI Reference Sequence: NM_017085.2) are marked in bold and highlighted in grey. An arrow indicates beginning of the translated region in the long form of aromatase. Untranslated regions have been grayed out. The 90 bp fragment (715–805) that was amplified in our qPCR and RT-PCR analyses is highlighted in yellow. The region that is thought to be missing in the truncated form of the enzyme involves the exons 2 and 3 and hence encompasses the sequence that was targeted in our study. The boxed nucleotides in the sequence indicate the start and the end of the RNA probe that protected only the long form of aromatase in ribonuclease protection assays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100628#pone.0100628-Roselli3" target="_blank">[10]</a>.</p

Cocaine increased ΔFosB protein levels in the dorsal striatum and nucleus accumbens.

<p>A) Representative western blots with three FosB antibodies: 5G4, sc-48, H-75. Due to its specific and robust labeling for ΔFosB isoforms (34–37 kD), 5G4 was used for subsequent experiments. B) Representative western blot of dorsal striatum samples from 2-week cocaine- or saline-treated males and females probed with 5G4 anti-FosB. The same blot reprobed for actin is shown below. Bar graph shows quantification of optical density from n = 3 animals/group. C) Representative western blot of nucleus accumbens samples from 2-week cocaine- or saline-treated rats probed with 5G4 anti-FosB. The same blot reprobed for actin is shown below. Quantification of optical density from n = 3 samples/group. Two-way ANOVAs showed that cocaine significantly increased ΔFosB in both dorsal striatum (B) and nucleus accumbens (C).</p

FosB immuno-staining in the striatum.

<p>Representative low magnification photomicrographs of striatal sections immuno-stained for FosB (5G4) from a saline- (A) and cocaine- (B) treated rat; sections were counter-stained with cresyl violet. C) Higher magnification photomicrograph from the dorsal striatum of a cocaine treated rat. FosB-IR nuclei of varying staining intensities that are in focus are indicated by arrows (dark = black arrow, medium = gray arrow, and light = white arrow); several cresyl violet-stained cells that are FosB-negative are indicated by open triangles. Scale bars: 100 µm for A and B, 10 µm for C.</p

Cocaine selectively increased the proportions of neurons darkly stained for FosB in the nucleus accumbens (NAc) Core of male and female rats.

<p>A) Bar graphs showing the percentage of all neurons in saline- (Sal) and cocaine- (Coc) treated male (M) and female (F) rats that were FosB-IR. Each bar shows the percentage of all neurons in each treatment group that were light, medium, or darkly stained (middle left, NAc Core), and darkly stained cells only (middle right, Dark). The partitioning scheme used to define dorsal-ventral (DV) and medial-lateral (ML) zones for anterior (left) and posterior (right) NAc Core are also shown. B and C) Bar graphs showing the proportion of FosB-IR cells in each anatomical zone of the anterior (B) and posterior (C) NAc Core. Each bar shows the percentage of all neurons in each treatment group that were light, medium, or darkly stained. The tables summarize statistically significant differences (p<0.05) for the NAc Core as a whole (A), and in the anterior (B) and posterior (C) anatomical zones. “D” indicates a statistically significant drug effect, and “S” indicates a significant sex difference (See text).</p

Cocaine increased locomotor activity to a greater extent in females than in males.

<p>A) Representative locomotor activity traces during the initial 5 min following saline (Sal) or cocaine (Coc) injection on the last day (Day 10) of treatment in males (M) and females (F). Blue lines represent the movement of each animal during this period. B) The mean±SEM distance traveled during 60 min following cocaine or saline injection each day over the 2 week treatment period. Cocaine increased locomotion, especially in female rats. C) Distance traveled on the first day (D1) and the average of week 2 (Wk2) for cocaine-treated male (left) and female (right) rats. Data are displayed separately for sensitizing and nonsensitizing rats; connected points represent data for the same individual. Nonsensitizing males showed consistently low activity throughout treatment, whereas nonsensitizing females showed consistently high activity throughout treatment. *p<0.05.</p

Heat maps of FosB-IR in the dorsal striatum.

<p>Numbers in the center indicate AP levels relative to Bregma. The cocaine-induced increase in FosB-IR is apparent at all levels in both male (left) and female (right) rats. Greater FosB-IR was seen in the medial, relative to the lateral, part of the dorsal striatum. Females exhibited higher FosB-IR than males, which is more apparent in the saline-treated rats. In addition, the cocaine-induced FosB-IR increase was larger in the more caudal (1.08 and 0.60) sections than in the rostral (1.56) section. Coc: cocaine, and Sal: saline.</p

Cocaine induced regionally specific increases in FosB- IR in the dorsal striatum of male and female rats.

<p>A) Bar graphs showing the percentage of all neurons in saline- (Sal) and cocaine- (Coc) treated male (M) and female (F) rats that were FosB-IR at any intensity level (Total FosB), and broken down into medium or darkly stained cells. B) Bar graphs showing the proportion of FosB-IR cells in each anatomical zone of the dorsal striatum. Each bar shows the percentage of all neurons in each treatment group that were light, medium, or darkly stained. Overall FosB-IR was higher in the Medial and Ventral, relative to the Lateral, zone, while cocaine-induced increases were most prominent in the Lateral zone. Additionally, females exhibited slightly higher FosB-IR than males. C) The partitioning scheme used to define Medial (M), Intermediate (I), Lateral (L), and Ventral (V) zones. D) A table summarizing the statistically significant differences (p<0.05) for dorsal striatum as a whole (Overall) and each of the zones. “D” indicates a significant drug effect, and “S” indicates a significant sex difference (See text).</p

Cocaine selectively increased the proportions of neurons darkly stained for FosB in the nucleus accumbens (NAc) Shell of male and female rats.

<p>A) A Bar graphs showing the percentage of all neurons in saline- (Sal) and cocaine- (Coc) treated male (M) and female (F) rats that were FosB-IR. Each bar shows the percentage of all neurons in each treatment group that were light, medium, or darkly stained (middle left, NAc Shell), and darkly stained cells only (middle right, Dark). The partitioning scheme used to define dorsomedial (DM) and ventrolateral (VL) zones for anterior (left) and posterior (right) NAc Shell are also shown. B and C) Bar graphs showing the proportion of FosB-IR cells in each anatomical zone of the anterior (B) and posterior (C) NAc Shell. Each bar shows the percentage of all neurons in each treatment group that were light, medium, or darkly stained. The tables summarize statistically significant differences (p<0.05) for the NAc Shell as a whole (A), in the anterior (B) and the posterior (C) anatomical zones. “D” indicates a statistically significant drug effect, and “S” indicates a significant sex difference (See text).</p